Liganddependent interaction of PPARδ with T cell …...1 Liganddependent interaction of PPARδ with T cell protein tyrosine phosphatase 45 enhances insulin signaling Taesik Yoo1,

1

Ligand-dependent interaction of PPARδ with T cell protein tyrosine

phosphatase 45 enhances insulin signaling

Taesik Yoo1, Sun Ah Ham

1, Won Jin Lee

1, Seon In Hwang

2, Jin-A Park

3, Jung Seok Hwang

1,

Jinwoo Hur1, Ho-Chul Shin

3, Sung Gu Han

1, Chi-Ho Lee

1, Dong Wook Han

2, Kyung Shin

Paek4, and Han Geuk Seo

1,5

1 Sanghuh College of Life Sciences, Konkuk University, Seoul, Korea;

2 Department of Stem Cell Biology, Konkuk University, Seoul, Korea;

3 Department of Veterinary Pharmacology and Toxicology, Konkuk University, Seoul, Korea;

4 Department of Nursing, Semyung University, Jechon, Korea.

5 Correspondence to:

Han Geuk Seo, Sanghuh College of Life Sciences, Konkuk University, 120 Neungdong-

ro, Gwangjin-Gu, Seoul 05029, Republic of Korea.

Tel.: +82-2-450-0428, Fax: +82-2-455-1044, E-mail: [email protected]

Running title: TCPTP45-mediated insulin sensitization of PPARδ

Page 1 of 55 Diabetes

Diabetes Publish Ahead of Print, published online December 12, 2017

2

ABSTRACT

Peroxisome proliferator-activated receptor (PPAR) δ plays a pivotal role in metabolic

homeostasis through its effect on insulin signaling. Although diverse genomic actions of

PPARδ are postulated, the specific molecular mechanisms whereby PPARδ controls insulin

signaling have not been fully elucidated. We demonstrate here that short-term activation of

PPARδ results in formation of a stable complex with nuclear T cell protein tyrosine

phosphatase 45 (TCPTP45) isoform. This interaction of PPARδ with TCPTP45 blocked

translocation of TCPTP45 into the cytoplasm, thereby preventing its interaction with the

insulin receptor, which inhibits insulin signaling. Interaction of PPARδ with TCPTP45

blunted interleukin 6-induced insulin resistance, leading to retention of TCPTP45 in the

nucleus, thereby facilitating deactivation of the STAT3-SOCS3 signal. Finally, GW501516-

activated PPARδ improved insulin signaling and glucose intolerance in high-fat diet-fed mice

through its interaction with TCPTP45. This novel interaction of PPARδ constitutes the most

upstream component identified of the mechanism downregulating insulin signaling.

Keywords: GW501516, insulin, PPARδ, protein-protein interaction, TCPTP45

Page 2 of 55Diabetes

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Insulin initiates its cellular actions by binding to its cell surface transmembrane receptor,

which is a tyrosine kinase (1). Upon binding of insulin to the extracellular α-subunit, the

intrinsic protein tyrosine kinase (PTK) activity of the intracellular β-subunit is activated,

leading to its autophosphorylation and subsequent phosphorylation of insulin receptor

substrate (IRS) proteins and other adaptor molecules (2). This tyrosine phosphorylation

provides docking sites for the recruitment of several src homology 2 domain-containing

proteins, such as the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K), which

mediate the biological activities of insulin in its target organs (2,3). Under the pathological

conditions associated with obesity and type 2 diabetes, impaired responses to insulin are

coupled with resistance to its actions in target organs, including liver, muscle, and adipose

tissue (3).

Evidence is emerging that protein tyrosine phosphatases (PTPs) play a pivotal role in

insulin receptor (IR) signaling, by dephosphorylating the IR within minutes of its activation,

to terminate IR-mediated insulin signaling (4,5). Both T cell protein tyrosine phosphatase

(TCPTP) and PTP1B are implicated as negative regulators of IR (4,6), but they have distinct

biological activities, by virtue of their substrate specificity and different subcellular locations

(4). TCPTP, which was originally identified in human T cells (7), has two alternative splice

variants: a 48 kDa form (TCPTP48), which contains a hydrophobic carboxy-terminus and is

localized to the endoplasmic reticulum (ER), similar to PTP1B, and a shorter 45 kDa form

(TCPTP45), which lacks the hydrophobic C-terminus and is targeted to the nucleus (8).

Although TCPTP45 is localized to the nucleus in resting cells, it is translocated to the

cytoplasm and plasma membrane in response to external stimuli such as insulin, where it can

access its substrates (9). Accordingly, TCPTP45 can dephosphorylate the IR in the cytoplasm

and at the plasma membrane, whereas ER-targeted TCPTP48 dephosphorylates the IR after it


4

has been endocytosed, similar to the action of PTP1B (5,9). In addition to utilizing receptor

PTKs, such as IR, as substrates, TCPTP also utilizes non-receptor PTKs as substrates,

including signal transducer and activator of transcription (STAT)-3 (10,11). Indeed, recent

studies demonstrated that STAT3, a target of TCPTP45, is implicated in the insulin resistance

induced by interleukin 6 (IL-6), through upregulation of suppressor of cytokine signaling 3

(SOCS3), which contributes to the negative regulation of insulin signaling by interacting with

the IR and IRS (12).

The biological activity of peroxisome proliferator-activated receptor (PPAR) δ, a nuclear

receptor, has been credited to transcriptional programs that regulate the expression of target

genes (the so-called genomic action) (13,14). However, there have been recent reports that

could not explain the biological functions of PPARδ by a genomic action alone (15,16). Here,

we report that short-term activation of PPARδ by high-affinity ligand results in the formation

of a stable complex with TCPTP45, thereby blocking the insulin-responsive translocation of

nuclear TCPTP45 into the cytoplasm, and preventing access of TCPTP45 to IR and its

inhibitory effect on insulin signaling. In addition, this protein-protein interaction blunts IL-6-

induced insulin resistance by accelerating TCPTP45-mediated deactivation of the STAT3-

SOCS3 signal, in a process mediated by sequestration of TCPTP45 within the nuclear

compartment. These observations demonstrate the role of a novel TCPTP45-mediated

pathway in the insulin sensitizing action of PPARδ.


5

RESEARCH DESIGN AND METHODS

Co-immunoprecipitation and immunoblot analysis

Cell and tissue lysates were prepared and then pre-cleared with protein G Sepharose. Pre-

cleared lysates were mixed with the specified antibodies and then precipitated with protein G

Sepharose. After washing with PBS, the immunoprecipitates and total lysates (input) were

subjected to immunoblot analysis.

Animal study

Male ICR mice (4-week-old, 15–20 g) were purchased from Orient Bio (Seongnam, Korea)

and maintained in pathogen-free environmental conditions on a 12 h light-dark cycle at 22 ±

2 °C. All procedures were approved by the Institutional Animal Care and Use Committee of

Konkuk University (approval number: KU16144). After 1 week of acclimation, the mice

were divided into two groups, and were fed either a normal diet (ND) (Altromin Spezialfutter

GmbH & Co., Lage, North Rhine-Westphalia, Germany) or a high-fat diet (HFD) (Research

Diets, Inc. NJ, USA) for 10 weeks, during which water and diet were available ad libitum.

Glucose and insulin tolerance tests

For intraperitoneal glucose tolerance testing (IPGTT), mice were fasted for 16 h, and then

glucose (2 g/kg body mass) was administered intraperitoneally. Blood was taken from the tail

vein at 0, 30, 60, 90, and 120 min after glucose injection, and the circulating glucose levels

were determined using a OneTouch automatic glucose monitor (LifeScan, Inc., Milpitas, CA,

USA). For intraperitoneal insulin tolerance testing (IPITT), insulin (0.5 units/kg of body mass)

in sterile saline was injected intraperitoneally, and the circulating glucose levels were

determined as described above.


6

Statistical analysis

Data are expressed as mean ± standard error (SE). Statistical significance was determined by

one-way ANOVA, followed by Tukey-Kramer or Student’s t-test, as appropriate.


7

RESULTS

PPARδ directly interacts with TCPTP45

In a yeast-hybrid assay that used PPARδ as bait, we identified a cDNA fragment encoding a

full-length amino acid sequence of TCPTP45 in a universal human cDNA library. The

interaction of PPARδ with TCPTP45 was confirmed by co-immunoprecipitation assays

performed in primary hepatocytes, hepatoblastoma-derived HepG2 cells, differentiated

C2C12 myotubes, and 3T3-L1 adipocytes. When cells were treated with GW501516, a

specific PPARδ agonist, immunoprecipitation of PPARδ from whole-cell lysates resulted in

the co-immunoprecipitation of TCPTP45. However, PTP1B, another tyrosine-specific

phosphatase that targets the insulin receptor β-subunit (IRβ), was not co-immunoprecipitated

with PPARδ (Fig. 1A–D). This GW501516-dependent interaction was almost completely

abolished in HepG2 cells stably expressing PPARδ small hairpin (sh)RNA, indicating that

ligand-dependent activation of PPARδ is a prerequisite for its interaction with TCPTP45 (Fig.

1E and Supplementary Fig. 1A). This interaction of PPARδ with TCPTP45 was also

confirmed using reciprocal antibodies for immunoprecipitation and immunoblotting (Fig. 1F).

No proteins were immunoprecipitated using control immunoglobulin G (IgG). Similar results

were obtained in HEK293T cells co-transfected with Flag-tagged PPARδ and hemagglutinin

(HA)-tagged TCPTP45, in which the GW501516-dependent interaction of PPARδ and

TCPTP45 was present at 30 min and reached a maximum at 45 min (Fig. 1G and

Supplementary Fig. 1B). In addition, when HEK293T cells were transfected with individual

plasmids expressing PPARα, δ, or γ, together with TCPTP45, in the presence or absence of

specific ligands for each PPAR isoform, PPARδ but not PPARα and γ demonstrated a strong


8

interaction with TCPTP45 in a co-immunoprecipitation assay, indicating that only the PPARδ

isoform can interact with TCPTP45 (Supplementary Fig.1C).

The bipartite nuclear localization sequence (NLS) of TCPTP45 is responsible for its

interaction with PPARδ

A direct interaction between PPARδ and TCPTP45 was further confirmed in glutathione S-

transferase (GST) pull-down assays, in which the majority of PPARδ was pulled down with

GST-fused TCPTP45 in the presence of GW501516 (Fig. 2A). Thus, we generated a panel of

TCPTP45 deletion mutants to identify the regions of TCPTP45 that are responsible for its

interaction with PPARδ (Fig. 2B). Whereas full-length TCPTP45 (FL) displayed a strong

interaction with PPARδ, the deletion mutants TCPTP45 (D1) and TCPTP45 (D2) showed no

significant interaction, indicating that the C-terminal region of TCPTP45 is essential for this

interaction (Fig. 2C). Because the C-terminal region of TCPTP45 has a bipartite nuclear

localization sequence (NLS), we constructed three NLS mutants to identify the critical amino

acid residue(s) responsible for the association of TCPTP45 with PPARδ. Site-directed

mutants were constructed by introducing amino acid substitutions in the N-terminal part of

NLS residues 350, 351, and 352 of TCPTP45 (TCPTP45350-A3

), and further substitutions in

the C-terminal part of NLS residues 378, 379, and 380 (TCPTP45378-A3

), and in both the N-

and C-terminal parts of NLS residues 350, 351, 352, 378, 379, and 380 (TCPTP45350/378-A3

),

replacing arginine or lysine residues with alanine (Fig. 2D).

When these mutant proteins were individually incubated with lysates prepared from

HEK293T cells that had been transfected with PPARδ, TCPTP45350-A3

displayed a weaker

ability than TCPTP45378-A3

to interact with PPARδ. Furthermore, the interaction between

PPARδ and TCPTP45 was nearly eliminated when PPARδ was incubated with


9

TCPTP45350/378-A3

, indicating that the bipartite NLS of TCPTP45 is indispensable for the

interaction with PPARδ (Fig. 2E). Consistent with this, the localization of TCPTP45 was

limited to the cytoplasm of HepG2 cells transfected with HA-tagged TCPTP45350/378-A3

(Fig.

2F).

Because TCPTP48 also has a bipartite NLS region, we examined the association

between PPARδ and TCPTP48 in HepG2 by co-immunoprecipitation. As expected, PPARδ

interacted with TCPTP48, suggesting that such protein-protein interactions via bipartite NLS

region may occur non-specifically under any conditions in which these molecules come into

close proximity by cellular destruction (Supplementary Fig. 2A). However, when the nuclear

or cytoplasmic fractions of HepG2 cells stimulated with GW501516 were analyzed to

confirm the specificity of TCPTP, an interaction between PPARδ and TCPTP was observed

in both fractions (Supplementary Fig. 2B and C). These results indicate that, although both

TCPTP45 and TCPTP48 can interact with PPARδ via their bipartite NLS, their subcellular

location is a unique determinant for a specific interaction with PPARδ.

To further evaluate the specificity of the bipartite NLS of TCPTP45 in the interaction

with PPARδ, the bipartite NLS of TCPTP45 was replaced with SV40 large T antigen NLS

(17). When the cells were transfected with TCPTP45350/378-SV40

, although the majority of

TCPTP45350/378-SV40

was observed in nuclear fraction, the interaction between PPARδ and

TCPTP45350/378-SV40

was not detected in the presence of GW501516 (Supplementary Fig. 2D-

F).

GW501516-activated PPARδ inhibits the insulin-triggered translocation of TCPTP45

from nucleus to cytoplasm

Because PPARδ interacted with both TCPTP45 and TCPTP48 in lysates prepared from


10

HepG2 cells, we examined the cellular co-localization of PPARδ and TCPTPs to confirm the

exact nature of the interaction with PPARδ. By confocal fluorescence microscopy using

fluorescent fusion protein versions of PPARδ and TCPTP45D182A

or TCPTP48D182A

, which

are substrate-trapping mutants that can form complexes with tyrosine-phosphorylated

substrates (18), we observed that green fluorescent protein (GFP)-tagged PPARδ was co-

localized with Discosoma sp. red fluorescent protein (DsRed)-tagged TCPTP45 in the

nucleus of Chinese Hamster Ovary cells stably expressing the insulin receptor (CHO-IR).

Upon stimulation with insulin, the majority of DsRed-TCPTP45 was rapidly translocated into

the cytoplasm, but this insulin-stimulated translocation was almost completely abolished in

cells treated with GW501516 (Supplementary Fig. 3A). By contrast, neither co-localization of

GFP-PPARδ and DsRed-TCPTP48, nor translocation between intracellular compartments,

was observed, stressing the importance of subcellular localization in the interaction between

PPARδ and TCPTP45 (Supplementary Fig. 3B).

To investigate whether GW501516-activated PPARδ affects the insulin-stimulated

interaction of TCPTP45 with IRβ, co-immunoprecipitation assays were performed using

Flag-PPARδ and HA-TCPTP45D182A

in CHO-IR and HepG2 cells. Upon stimulation with

insulin, IRβ was co-immunoprecipitated with TCPTP45D182A

in both cells, whereas an

interaction between PPARδ and TCPTP45D182A

was not observed in the absence of

GW501516. By contrast, insulin-stimulated complex formation between IRβ and

TCPTP45D182A

was reduced in cells treated with GW501516, because of increased interaction

between PPARδ and TCPTP45D182A

. Furthermore, GSK0660-mediated antagonism of

GW501516 caused the dissociation of PPARδ and TCPTP45D182A

, with a concomitant

increase in the formation of IRβ and TCPTP45D182A

complexes (Fig. 3A and B). Similar

results were obtained from HepG2 cells stably expressing PPARδ or control shRNA (Fig.


11

3C). As expected, insulin-stimulated translocation of TCPTP45D182A

from the nucleus to the

cytoplasm was almost completely abolished in the presence of GW501516 with a

concomitant reduction in the interaction of TCPTP45D182A

and phosphorylated IRβ, and this

GW501516-mediated inhibition of TCPTP45D182A

translocation and interaction with IRβ was

prevented by GSK0660 (Fig. 3D).

The GW501516-stimulated interaction of PPARδ with TCPTP45 potentiates the insulin

signal

Because GW501516 induced an interaction between PPARδ and TCPTP45, particular

attention was paid to the effect on insulin signaling, because TCPTP45 can antagonize insulin

signaling by dephosphorylating IRβ (5). When HepG2 cells were treated with insulin, IRβ

was rapidly phosphorylated, and this effect was further potentiated in the presence of

GW501516 (Fig. 4A). This GW501516-mediated potentiation of IRβ phosphorylation was

almost completely abolished in HepG2 cells transfected with TCPTP45350/378-A3

, a site-

directed mutant lacking the molecular regions that interact with PPARδ (Fig. 4B). Consistent

with these results, GW501516-mediated potentiation of IRβ phosphorylation was also

manifested in cells that transiently expressed TCPTP45, but not TCPTP48 (Fig. 4C).

Furthermore, small interfering (si)RNA-mediated silencing of TCPTP, but not PTP1B,

prevented the effect of GW501516 to potentiate IRβ phosphorylation (Fig. 4D and

Supplementary Fig. 4A). Similar results are shown in primary hepatocytes (Fig. 4E).

GW501516-activated PPARδ extends the duration of insulin-stimulated IRβ

phosphorylation

When HepG2 cells were exposed to insulin, IRβ phosphorylation peaked after 2–5 min, and


12

then rapidly returned to basal levels. However, pretreatment with GW501516 for 30 min

sustained IRβ phosphorylation until 60 min after the initial insulin exposure (Fig. 4F and H).

This effect of GW501516 on the duration of IRβ phosphorylation was further potentiated in

cells ectopically expressing PPARδ (Fig. 4G and H, and Supplementary Fig. 4B and C). This

effect of GW501516 remained significant for as long as 6 h in cells expressing PPARδ

(Supplementary Fig. 4D). Although this effect of GW501516 on IRβ phosphorylation was

prevented by shRNA-mediated silencing of PPARδ (Fig. 4I-K), it was rescued by ectopic

overexpression of PPARδ in transfectants stably expressing PPARδ shRNA (Fig. 4L-N). By

contrast, treatment with WY14643 or rosiglitazone, specific ligands for PPARα and PPARγ,

respectively, did not affect insulin-stimulated phosphorylation of IRβ (Supplementary Fig. 4E

and 4F).

GW501516-activated PPARδ regulates IRβ phosphorylation in a TCPTP45-dependent

manner

Ectopic expression of TCPTP45 inhibited the effect of GW501516 on IRβ phosphorylation in

cells stably expressing PPARδ shRNA but not control shRNA, while this effect of

GW501516 was rescued by ectopic expression of PPARδ (Fig. 5A and Supplementary Fig.

5A). Furthermore, The GW501516-stimulated interaction between PPARδ and TCPTP45

demonstrated in PPARδ precipitates was clearly correlated with increased phosphorylation of

IRβ (Fig. 5B). However, this effect of GW501516 was almost completely abolished in the co-

immunoprecipitate of TCPTP45350/378-A3

, indicating that the GW501516-stimulated

interaction of PPARδ with TCPTP45 prevents the access of TCPTP45 to cytoplasmic

tyrosine-phosphorylated IRβ (Fig. 5C).


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The GW501516-stimulated interaction of PPARδ with TCPTP45 influences downstream

IR signaling

IR signaling has its biological effects in target organs through phosphorylation of intracellular

effector molecules such as IRS-1 and Akt (2). Consistent with the effect of GW501516 on

IRβ phosphorylation, GW501516 treatment prolonged the duration of insulin-stimulated IRS-

1 and Akt phosphorylation for 6 h in HepG2 cells stably expressing control shRNA

(Supplementary Fig. 5B), while these effects of GW501516 were dramatically inhibited in the

presence of PPARδ shRNA (Fig. 5D). In addition, the GW501516-stimulated interaction of

PPARδ with TCPTP45 observed in PPARδ precipitates was correlated with the increased

phosphorylation of IRS-1 and Akt, while these effects of GW501516 were almost completely

abolished when TCPTP45350/378-A3

was co-immunoprecipitated, indicating that the interaction

between PPARδ and TCPTP45 directly correlates with IRS-1 and Akt phosphorylation (Fig.

5E).

We next assessed glucose levels in the HepG2 cells, to evaluate whether GW501516-

mediated activation of PPARδ is directly coupled to the biological effects of insulin. Upon

exposure to insulin for 30 min, glucose levels in the glucose production medium dramatically

decreased, and this reduction in glucose output was further enhanced by treatment with

GW501516 in HepG2 cells stably expressing control shRNA. However, this effect of

GW501516 on glucose levels in the glucose production medium was significantly inhibited in

cells stably expressing PPARδ shRNA (Fig. 5F).

GW501516-activated PPARδ regulates insulin signaling in myotubes and adipocytes

Because skeletal muscle and adipocytes are also major target organs of insulin action (1), we

investigated the effect of GW501516 on insulin signaling in differentiated mouse C2C12


14

myotubes and 3T3-L1 adipocytes. Consistent with the results observed in HepG2 cells,

GW501516 also increased the duration of IRβ and Akt phosphorylation by promoting

interaction between PPARδ and TCPTP45 (Fig. 6A and B, and Supplementary Fig. 6A and

B). Similar results were also observed in C2C12 myotubes transfected with epitope-tagged

PPARδ and TCPTP45, while the effects of GW501516 on the interaction of PPARδ with

TCPTP45 and insulin signaling were almost completely abolished in C2C12 myotubes

transfected with epitope-tagged PPARδ and TCPTP45350/378-A3

(Supplementary Fig. 6C).

Furthermore, insulin-induced translocation of Glut4 to the plasma membrane was sustained

for 6 h in cells treated with both insulin and GW501516, whereas the Glut4 signal was

predominantly cytoplasmic in cells treated with insulin alone at this time point (Fig. 6C and

D).

The GW501516-stimulated interaction between PPARδ and TCPTP45 mitigates IL-6-

induced insulin resistance

Since obesity-associated chronic inflammation of adipose tissue leads to insulin resistance in

peripheral tissues, including liver, and TCPTP45 is implicated in glucose homeostasis

through STAT3 signaling in liver (10,19), we examined the effects of GW501516 on IL-6-

induced insulin resistance. Insulin enhanced the phosphorylation of IRS-1 and Akt in HepG2

cells, but the insulin-stimulated phosphorylation of IRS-1 and Akt was markedly attenuated

in the presence of IL-6. However, this IL-6-mediated suppression of IRS-1 and Akt

phosphorylation was reversed by GW501516, implying a possible role for PPARδ in the

antagonism of inflammation-induced insulin resistance (Fig. 6E).

Because STAT3 and SOCS3 are implicated in IL-6-induced insulin resistance (12,20),

we examined the effects of GW501516 on the activation and expression of STAT3 and


15

SOCS3. Although insulin-stimulated phosphorylation of STAT3 and expression of SOCS3

were inhibited in HepG2 cells stably expressing control shRNA in the presence of

GW501516, these effects of GW501516 were substantially prevented by the expression of

PPARδ shRNA (Fig. 6F). Consistent with these results, siRNA-mediated knockdown of

SOCS3 prevented the IL-6-triggered suppression in the phosphorylation of IRS-1 and Akt,

indicating that SOCS3 plays a role in the GW501516-dependent modulation of insulin

resistance induced by IL-6 (Supplementary Fig. 6D). In addition, GW501516-stimulated

interaction between PPARδ and TCPTP45 observed in PPARδ precipitates was directly

correlated with the increased phosphorylation of Akt, with concomitant reductions in the

levels of STAT3 phosphorylation and SOCS3 expression. However, these effects of

GW501516 were significantly blunted in cells transfected with TCPTP45350/378-A3

instead of

TCPTP45, indicating that the GW501516-stimulated interaction of PPARδ with TCPTP45

improves IL-6-induced insulin resistance by targeting STAT3 and SOCS3 (Fig. 6G). Similar

results were also observed in primary hepatocytes (Fig. 6H).

Administration of GW501516 enhances insulin signaling in animal models

We next wished to evaluate whether GW501516-activated PPARδ can affect glucose

metabolism in ND- or HFD-fed mice. In follow-up experiments, we fed mice a ND or HFD

for 10 weeks and checked their body masses weekly to confirm the expected effect of HFD

feeding (Supplementary Fig. 7A). When mice were administered with GW501516 or DMSO

and challenged 30 min later with a bolus of glucose by intraperitoneal injection, blood

glucose levels of ND- or HFD-fed mice rapidly increased, to peak 30 min later. However,

blood glucose subsequently decreased more rapidly in HFD-fed mice that had been

administered with GW501516 than in those that had not (Fig. 7A). Analogous results were


16

obtained in HFD-fed mice subjected to IPITT, in which the insulin-stimulated decrease in

blood glucose was greater following GW501516 administration (Fig. 7B).

To determine whether the administration of GW501516 directly affected the interaction

between PPARδ and TCPTP45, leading to an improvement of insulin signaling in vivo, we

analyzed the interaction by co-immunoprecipitation in lysates prepared from liver, skeletal

muscle, and adipose tissue. The interaction between PPARδ and TCPTP45 was greatest in the

tissues of ND-fed mice administered with GW501516, with a lesser effect of GW501516

being detected in HFD-fed mouse tissues. This GW501516-mediated enhancement in the

PPARδ/TCPTP45 interaction was closely correlated with the phosphorylation levels of IRβ

and Akt in these tissues (Fig. 7C-E). Interestingly, the circulating levels of insulin, glucose,

and IL-6 were significantly higher in HFD-fed mice than in ND-fed mice, but the levels of

insulin and glucose were significantly lower in mice treated with GW501516 for 30 min (Fig.

7F and G, and Supplementary Fig. 7C), implying an important role for PPARδ in insulin

action and whole body glucose homeostasis in vivo.


17

DISCUSSION

Short-term activation of PPARδ modulates IR signaling by preventing translocation of the IR-

specific phosphatase TCPTP45 into the cytoplasm because of their direct protein-protein

interaction. Unlike the genomic action of the transcription factor PPARδ, little is known

about non-genomic actions of PPARδ, such as its protein-protein interactions. Based on the

concept that PPARδ exerts its biological actions by regulating the transcription of target genes

(13), recent studies demonstrated that ligand-activated PPARδ improves insulin signaling

through transcriptional regulation of target genes such as insulin-induced gene-1, pyruvate

dehydrogenase kinase 4, and angiopoietin-like protein 4 in hepatocytes and myotubes (21,22).

In the most recent reports, cells were treated with specific PPARδ ligands for as long as

18 to 24 h, so that the effect of PPARδ activation on transcriptional programs and

downstream biological activities could be investigated (23,24). However, we show herein that

the activation of PPARδ by GW501516 for a period of only 30 min was sufficient to

phosphorylate IRβ and consequently IRS-1 and Akt. To achieve this effect, GW501516

promoted the interaction of PPARδ with TCPTP45, but could not affect the transcription of

TCPTP45. These findings suggest that non-genomic actions of PPARδ, rather than its

genomic actions, are important for the regulation of IR signaling. Thus, we propose a novel

mechanism whereby PPARδ influences insulin signaling, involving the sequestration of

PPARδ in the nucleus secondary to a protein-protein interaction with the IRβ-specific

phosphatase TCPTP45, therefore preventing binding to its cytoplasmic substrate IRβ.

Although GW501516-activated PPARδ interacted with both TCPTP45 and TCPTP48, it

co-localized in the nucleus with TCPTP45, but not TCPTP48. These two alternative splice

variants exhibit distinct subcellular localizations because of a difference in the seven amino


18

acid residues at their carboxy-terminals. As a result, TCPTP48 is mainly localized to the ER,

while TCPTP45 is restricted to the nucleus in resting cells (9). Therefore, TCPTP48

dephosphorylates IRβ after it is endocytosed into the ER in response to insulin, whereas

TCPTP45 dephosphorylates IRβ in the cytoplasm and at the plasma membrane after its

translocation from the nucleus (6,9). In this context, the PPARδ-mediated prevention of

TCPTP45 translocation, but not that of TCPTP48, into the cytoplasm may contribute to the

enhancement in insulin signaling. GW501516-activated PPARδ also did not interact with

PTP1B, another tyrosine-specific phosphatase that targets IRβ in the ER (4,6). Thus, the

subcellular localization, as well as the other biochemical properties of the substrate, when

interacting with PPARδ, may be critical determinants of the effects of PPARδ on insulin

signaling.

PPARδ, but not PPARα or γ, specifically interacted with TCPTP45 in a ligand-

dependent manner to enhance insulin signaling. Previous studies demonstrated that specific

ligands for PPARγ enhance insulin signaling through genomic actions regulating gene

transcription, including that of adipocyte P2 (aP2), lipoprotein lipase (LPL), fatty acid-

transport protein (FATP), Glut4, glucokinase, uncoupling protein (UCP)-2 and UCP-3, to

regulate glucose homeostasis, adipocyte differentiation, and lipid storage in cell or animal

models (25,26). In addition, ligand-activated PPARα also ameliorated insulin resistance by

upregulating genes encoding FATP, acyl-CoA oxidase, and LPL, leading to increased hepatic

fatty acid uptake and lipid metabolism (27,28). These findings are consistent with the data

reported here, because both enhance insulin signaling, but the underlying mechanisms

mediating this effect are PPAR isoform-specific. Indeed, only a 30 min treatment with

GW501516 was sufficient to activate PPARδ and enhance insulin signaling by inhibiting

translocation of the IRβ-specific phosphatase TCPTP45 into the cytoplasm. This was the


19

result of a direct interaction with TCPTP45, which we term a non-genomic action here.

Consequently, the effect of each PPAR nuclear receptor on insulin signaling can be

characterized according to its genomic and/or non-genomic actions.

Although the present findings are consistent with previous reports that GW501516-

activated PPARδ ameliorates IL-6-induced insulin resistance in hepatocytes by inhibiting the

STAT3-SOCS3 pathway (29), the molecular mechanisms may differ between studies, because

30 min treatment with GW501516 was sufficient to inactivate the STAT3-SOCS3 pathway in

this study. Several recent studies demonstrated that activation of PPARδ by specific ligands

alleviated IL-6-induced insulin resistance by suppressing SOCS3 expression, secondary to

inhibition of STAT3 activation. These effects of GW501516 were observed in hepatocytes

and adipocytes treated for 24 h, suggesting that the effects of PPARδ in this context may be

principally dependent on its genomic activity (30,31). Indeed, TCPTP45 was reported to

inhibit IL-6-induced STAT3 phosphorylation, leading to the attenuation of insulin signaling in

the liver (10,11). Consistent with these reports, PPARδ-mediated retention of TCPTP45 in the

nucleus facilitated the access of TCPTP45 to its substrate STAT3, resulting in reduced IL-6-

mediated phosphorylation and suppression of STAT3-mediated SOCS3 expression, which is

linked to insulin resistance (19,29). Thus, the insulin sensitizing effects of GW501516 may

be collectively mediated by the interaction between PPARδ and TCPTP45.

As opposed to our present findings showing TCPTP45-dependent effect of PPARδ in

insulin signaling, a previous study demonstrated that TCPTP deficiency in muscle has no

effect on insulin signaling and glucose homeostasis (32). Although this discrepancy for

TCPTP action in muscle insulin signaling is not fully elucidated at present, our present data

evenly showed that GW501516-dependent interaction of PPARδ and TCPTP45 potentiated

the insulin-triggered phosphorylation of IRβ and Akt and translocation of Glut4 in C2C12


20

myotubes. In fact, previous studies reported that ligand-activated PPARδ ameliorates cellular

energy homeostasis through its genomic action regulating transcription of genes including

UCP-2, UCP-3, pyruvate dehydrogenase kinase 4, carnitine palmitoyltransferase 1, and

malonyl-CoA decarboxylase in skeletal muscle (13,22-25). Thus, the possibility remains that

the effects of PPARδ activation on the insulin signaling may be attributed to their

transcriptional regulation of multiple genes rather than to direct interaction of PPARδ and

TCPTP45 in C2C12 myotubes. Further studies are necessary to clarify the exact molecule(s)

associated with PPARδ-mediated insulin signaling in muscle.

Consistent with the in vitro data, mice exposed to GW501516 for as little as 30 min

showed enhanced insulin-stimulated phosphorylation of IRβ and Akt in liver, skeletal muscle,

and adipose tissue. This GW501516-mediated enhancement in insulin signaling and glucose

homeostasis, which occurred even in HFD-fed obese mice, was accompanied by greater

interaction between PPARδ and TCPTP45, indicating that these in vivo effects of GW501516

are dependent on the association between PPARδ with TCPTP45, as in the cell models. To

date, a number of studies have shown that activation of PPARδ regulates insulin signaling by

regulating the expression of genes involved in lipid and glucose homeostasis. However, the

present study implies that PPARδ has multiple modes of action, and can enhance insulin

signaling through an interaction with TCPTP45, which we characterized as a non-genomic

action. These findings may suggest novel alternative future therapeutic strategies for

disorders related to defective insulin signaling.


21

Acknowledgments

Funding. This work was supported in part by the Basic Science Research Program through

the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT

and future Planning (NRF-2017R1A2A2A05001249 and 2015R1A5A1009701), and by the

Next-Generation BioGreen 21 Program (no. PJ01122201), Rural Development

Administration, Republic of Korea.

Conflict of Interest statement. No potential conflicts of interest relevant to this article

were reported.

Author contributions. T.Y. and H.G.S. conceived and designed the experiments; T.Y.,

S.A.H., W.J.L., S.I.H., J.P., J.S.H., J.H., H.S., S.G.H., C.L., and D.W.H. performed the

experiments; T.Y., K.S.P., and H.G.S. analyzed the results and wrote the manuscript. T.Y and

H.G.S is the guarantor of this work and, as such, had full access to all the data in the study

and takes responsibility for the integrity of the data and the accuracy of the data analysis.


22

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24

FIGURE LEGENDS

Figure 1―PPARδ directly interacts with TCPTP45. Primary hepatocytes (A), HepG2 cells

(B), C2C12 myotubes (C), or 3T3-L1 adipocytes (D) were treated with 100 nM GW501516

or DMSO for 45 min, and then whole-cell lysates were prepared and immunoprecipitated

with IgG or anti-PPARδ antibody. The immunoprecipitates and total lysates (input) were

subjected to immunoblot analysis with specific antibodies to detect the indicated proteins.

Two percent of each whole-cell lysate was used as the input. E: HepG2 cells stably

expressing control shRNA or shRNA targeting PPARδ were treated with 100 nM GW501516

or DMSO for 45 min. Whole-cell lysates were immunoprecipitated with IgG or anti-PPARδ

antibody and analyzed by immunoblotting. F: HepG2 cells treated with 100 nM GW501516

or DMSO for 45 min were lysed and immunoprecipitated with IgG or anti-TCPTP45

antibody. The immunoprecipitates and total lysates (input) were subjected to immunoblot

analysis. G: HEK293T cells co-transfected with Flag-hPPARδ and/or HA-hTCPTP45 for 48

h were treated with 100 nM GW501516 or DMSO for 45 min, and then whole-cell lysates

were prepared and immunoprecipitated with IgG or anti-Flag antibody.

Figure 2―PPARδ targets the bipartite nuclear localization signal of TCPTP45. Whole-cell

lysates prepared from HEK293T cells transfected with Flag-tagged hPPARδ for 48 h were

incubated with recombinant GST or GST-hTCPTP45 fusion protein (A), GST or GST-fused

deletion mutants (C), or GST or GST-fused site-directed mutants (E) immobilized to

glutathione-Sepharose 4B beads for 20 h in the presence of 100 nM GW501516 or DMSO.

Bead-bound proteins were analyzed by immunoblotting. Two percent of each whole-cell

lysate was used as the input. B: Schematic illustrations of GST-hTCPTP45 fusion protein


25

constructs. D: Schematic illustration of the C-terminal bipartite NLS region of TCPTP45. F:

HepG2 cells were transfected with pcDNA-HA-hTCPTP45350/378-A3

for 48 h and then

separated into nuclear (N) and cytoplasmic (C) fractions. Each fraction was then subjected to

immunoblot analysis.

Figure 3―GW501516-activated PPARδ inhibits insulin-stimulated translocation of

TCPTP45 from the nucleus to the cytoplasm. A and B: CHO-IR (A) or HepG2 cells (B) co-

transfected with Flag-PPARδ and HA-hTCPTP45D182A

for 48 h were treated with 1 µM

GSK0660 or DMSO for 30 min, and then treated with 100 nM GW501516. After a further 30

min incubation, the cells were exposed to 100 nM insulin for a final 30 min. C: HepG2 cells

stably expressing PPARδ or control shRNA were transfected with HA-hTCPTP45D182A

for 48

h and then treated with 100 nM GW501516 for 30 min. Following exposure 100 nM insulin

for a final 30 min, whole-cell lysates were immunoprecipitated and analyzed by immunoblot

analysis. Two percent of each whole-cell lysate was used as the input. D: CHO-IR cells co-

transfected with GFP-PPARδ and DsRed-hTCPTP45D182A

for 48 h were incubated with 1 µM

GSK0660 or DMSO for 30 min, and then treated with 100 nM GW501516 for 30 min

incubation. After exposure to 100 nM insulin for a final 30 min, the co-localization of

TCPTP45 with PPARδ or phosphorylated IRβ is indicated by the presence of yellow or pink

in the merged image, respectively. For phosphorylated IRβ, the fixed cells were sequentially

reacted with primary anti-phospho-IRβ antibody and Alexa Fluor 680-conjugated secondary

antibody, and then the fluorescent signals were visualized. Scale bar = 20 µm.

Figure 4―GW501516-activated PPARδ extends the duration of insulin-stimulated IRβ

phosphorylation. A: HepG2 cells were treated with 100 nM GW501516 or DMSO for 30 min,


26

and then treated with or without 100 nM insulin for a further 30 min. Whole-cell lysates were

prepared and subjected to immunoblot analysis. HepG2 cells co-transfected with HA-tagged

plasmids expressing hTCPTP45 or hTCPTP45350/378-A3

(B), or hTCPTP45 or hTCPTP48 (C),

for 48 h were treated with 100 nM GW501516 or DMSO for 30 min, and then exposed to

insulin for a further 30 min. Whole-cell lysates were prepared and subjected to immunoblot

analysis. D and E: HepG2 cells (D) or primary hepatocytes (E) were transfected with or

without control siRNA or siRNA targeting hTCPTP for 48 h, and then treated with 100 nM

GW501516 or DMSO. After incubation for 30 min, the cells were treated with or without 100

nM insulin for a further 30 min. Whole-cell lysates were analyzed by immunoblotting. F:

HepG2 Cells were treated with 100 nM GW501516 or DMSO for 30 min, and then exposed

to 100 nM insulin for the indicated periods of time. G: HepG2 cells transfected with Flag-

hPPARδ for 48 h were treated with 100 nM GW501516 or DMSO for 30 min, and then

exposed to 100 nM insulin for the indicated periods of time. H: Immunoblot analyses of (F)

and (G) were quantified and represented with DMSO (black circles), GW501516 (white

circles), Flag-hPPARδ overexpression + DMSO (black triangles) and Flag-hPPARδ

overexpression + GW501516 (white triangles). Data are expressed as mean ± SE (n = 3). *p <

0.01 vs DMSO-treated group. #p < 0.01 vs DMSO-treated Flag-hPPARδ group. I-K: HepG2

cells stably expressing control shRNA (I) or shRNA targeting PPARδ (J) were treated with

100 nM GW501516 or DMSO for 30 min, and then exposed to 100 nM insulin for the

indicated periods of time. The immunoblots were then quantified and represented with

control shRNA + DMSO (black circles), control shRNA + GW501516 (white circles),

hPPARδ shRNA + DMSO (black triangles) and hPPARδ shRNA + GW501516 (white

triangles) (K). Results are expressed as mean ± SE (n = 3). *p < 0.01 vs DMSO-treated

control shRNA group. L-N: HepG2 cells stably expressing control shRNA (L) or shRNA


27

targeting PPARδ (M) were transfected with either pcDNA-Flag or pcDNA-Flag-hPPARδ for

48 h. The cells were treated with 100 nM GW501516 or DMSO for 30 min, and then exposed

to 100 nM insulin for a further 30 min. Immunoblots were then quantified and represented

with control shRNA (red bars) or hPPARδ shRNA (blue bars) (N). Results are expressed as

mean ± SE (n = 3). **, ##

p < 0.05.

Figure 5―The GW501516-stimulated interaction of PPARδ with TCPTP45 directly affects

the insulin receptor. A: HepG2 cells stably expressing PPARδ shRNA were co-transfected

with pcDNA-HA, pcDNA-HA-hTCPTP45, pcDNA-Flag, and/or pcDNA-Flag-hPPARδ for

48 h. The cells were treated with 100 nM GW501516 or DMSO for 30 min, and then exposed

to 100 nM insulin for a further 30 min. Whole-cell lysates were analyzed by immunoblotting.

B and C: HepG2 cells co-transfected with pcDNA-Flag-hPPARδ and pcDNA-HA-hTCPTP45

(B) or pcDNA-HA-hTCPTP45350/378-A3

(C) for 48 h were treated with 100 nM GW501516 or

DMSO for 30 min, and then exposed to 100 nM insulin for a further 30 min. Cell lysates

were then immunoprecipitated with anti-Flag antibody and analyzed by immunoblotting. D:

HepG2 cells stably expressing PPARδ shRNA were treated with 100 nM GW501516 or

DMSO for 30 min, and then exposed to 100 nM insulin for the indicated periods of time.

Whole-cell lysates were analyzed by immunoblotting. E: HepG2 cells co-transfected with

pcDNA-Flag-hPPARδ and pcDNA-HA-hTCPTP45 or pcDNA-HA-hTCPTP45350/378-A3

for 48

h were treated with 100 nM GW501516 or DMSO for 30 min, and then exposed to 100 nM

insulin for a further 6 h. Whole-cell lysates were immunoprecipitated and analyzed by

immunoblotting. F: HepG2 cells stably expressing shRNA against control or PPARδ were

treated with 100 nM GW501516 or DMSO for 30 min. Following exposure to 100 nM

insulin for 8 h, the quantity of glucose present in the glucose production medium was


28

determined and represented with control shRNA (red bars) or hPPARδ shRNA (blue bars).

Results are expressed as mean ± SE (n = 3). **p < 0.05.

Figure 6―GW501516-activated PPARδ modulates downstream insulin sensitivity and

resistance through its interaction with TCPTP45. Differentiated C2C12 myotubes (A and C)

or 3T3-L1 adipocytes (B and D) were treated with 100 nM GW501516 or DMSO for 30 min

and then exposed to 100 nM insulin for the indicated periods of time. The cells were

subjected to immunoblot (A and B) or fluorescence image analysis (B and D). For Glut4

translocation assay, the fixed cells were sequentially reacted with primary anti-Glut4 antibody

and Alexa Fluor 488-conjugated secondary antibody, and then the fluorescent signals were

visualized. Nuclei were stained with PI. Scale bars = 20 µm. E: HepG2 cells treated with or

without 20 ng/ml IL-6 for 90 min were incubated with 100 nM GW501516 or DMSO for 30

min, and then exposed to 100 nM insulin for a further 30 min. The cells were lysed and

analyzed by immunoblotting. F: HepG2 cells stably expressing control shRNA or shRNA

targeting PPARδ were treated with or without 20 ng/ml IL-6 for 90 min. The cells were then

incubated with 100 nM GW501516 or DMSO for a further 30 min, and then exposed to 100

nM insulin for an additional 30 min. Whole-cell lysates were then analyzed by

immunoblotting. G and H: HepG2 cells co-transfected with pcDNA-Flag-hPPARδ and

pcDNA-HA-hTCPTP45 or pcDNA-HA-hTCPTP45350/378A3

(G), or primary hepatocytes (H)

were treated with or without 20 ng/ml IL-6 for 90 min. The cells were then incubated with

100 nM GW501516 or DMSO for a further 30 min, and then exposed to 100 nM insulin for a

final 30 min. Whole-cell lysates were immunoprecipitated and analyzed by immunoblotting.

Two percent of each whole-cell lysate was used as the input.


29

Figure 7―Administration of GW501516 improves in vivo insulin sensitivity and glucose

tolerance through its interaction with TCPTP45. Mice fed a ND or HFD for 10 weeks were

fasted for 14 h, and treated with 10 mg/kg GW501516 or DMSO for 30 min. Following an

intraperitoneal injection of 2 g/kg glucose or 0.5 units/kg insulin, glucose (A) or insulin (B)

tolerance tests were performed. Blood glucose was then measured and represented with ND +

DMSO (black circles), ND + GW501516 (white circles), HFD + DMSO (black triangles) and

HFD + GW501516 (white triangles). Data are expressed as mean ± SE (n = 6). *p < 0.01 vs

HFD + DMSO group. Mice fed a ND or HFD for 10 weeks were fasted for 14 h, and then

treated with 10 mg/kg GW501516 or DMSO for 30 min. Following exposure to 0.5 units/kg

insulin for 30 min, the mice were sacrificed by inhalation of CO2 gas, and then liver (C),

skeletal muscle (D), and adipose tissue (E) were removed. Tissue homogenates were prepared

and immunoprecipitated with anti-TCPTP45 antibody. The total lysates (input) and

immunoprecipitates were analyzed by immunoblotting. Two percent of each tissue lysate was

used as the input. Mice were fed a ND or HFD for 10 weeks and then starved for 14 h. They

were then treated with 10 mg/kg GW501516 (red bars) or DMSO (blue bars) for 30 min and

sacrificed. Circulating levels of insulin and glucose were determined by ELISA or

spectrometric methods using sera prepared from blood collected from the carotid artery. Data

are expressed as mean ± SE (n = 6). *, #p < 0.01,

##p < 0.05.


Figure 1―PPARδ directly interacts with TCPTP45. Primary hepatocytes (A), HepG2 cells (B), C2C12 myotubes (C), or 3T3-L1 adipocytes (D) were treated with 100 nM GW501516 or DMSO for 45 min, and

then whole-cell lysates were prepared and immunoprecipitated with IgG or anti-PPARδ antibody. The

immunoprecipitates and total lysates (input) were subjected to immunoblot analysis with specific antibodies to detect the indicated proteins. Two percent of each whole-cell lysate was used as the input. E: HepG2 cells stably expressing control shRNA or shRNA targeting PPARδ were treated with 100 nM GW501516 or DMSO for 45 min. Whole-cell lysates were immunoprecipitated with IgG or anti-PPARδ antibody and analyzed by

immunoblotting. F: HepG2 cells treated with 100 nM GW501516 or DMSO for 45 min were lysed and immunoprecipitated with IgG or anti-TCPTP45 antibody. The immunoprecipitates and total lysates (input)

were subjected to immunoblot analysis. G: HEK293T cells co-transfected with Flag-hPPARδ and/or HA-hTCPTP45 for 48 h were treated with 100 nM GW501516 or DMSO for 45 min, and then whole-cell lysates

were prepared and immunoprecipitated with IgG or anti-Flag antibody.

190x142mm (300 x 300 DPI)


Figure 2―PPARδ targets the bipartite nuclear localization signal of TCPTP45. Whole-cell lysates prepared from HEK293T cells transfected with Flag-tagged hPPARδ for 48 h were incubated with recombinant GST or

GST-hTCPTP45 fusion protein (A), GST or GST-fused deletion mutants (C), or GST or GST-fused site-

directed mutants (E) immobilized to glutathione-Sepharose 4B beads for 20 h in the presence of 100 nM GW501516 or DMSO. Bead-bound proteins were analyzed by immunoblotting. Two percent of each whole-cell lysate was used as the input. B: Schematic illustrations of GST-hTCPTP45 fusion protein constructs. D: Schematic illustration of the C-terminal bipartite NLS region of TCPTP45. F: HepG2 cells were transfected with pcDNA-HA-hTCPTP45350/378-A3 for 48 h and then separated into nuclear (N) and cytoplasmic (C)

fractions. Each fraction was then subjected to immunoblot analysis.

135x101mm (300 x 300 DPI)


Figure 3―GW501516-activated PPARδ inhibits insulin-stimulated translocation of TCPTP45 from the nucleus to the cytoplasm. A and B: CHO-IR (A) or HepG2 cells (B) co-transfected with Flag-PPARδ and HA-

hTCPTP45D182A for 48 h were treated with 1 µM GSK0660 or DMSO for 30 min, and then treated with 100

nM GW501516. After a further 30 min incubation, the cells were exposed to 100 nM insulin for a final 30 min. C: HepG2 cells stably expressing PPARδ or control shRNA were transfected with HA-hTCPTP45D182A for 48 h and then treated with 100 nM GW501516 for 30 min. Following exposure 100 nM insulin for a final 30 min, whole-cell lysates were immunoprecipitated and analyzed by immunoblot analysis. Two percent of each whole-cell lysate was used as the input. D: CHO-IR cells co-transfected with GFP-PPARδ and DsRed-hTCPTP45D182A for 48 h were incubated with 1 µM GSK0660 or DMSO for 30 min, and then treated with

100 nM GW501516 for 30 min incubation. After exposure to 100 nM insulin for a final 30 min, the co-localization of TCPTP45 with PPARδ or phosphorylated IRβ is indicated by the presence of yellow or pink in

the merged image, respectively. For phosphorylated IRβ, the fixed cells were sequentially reacted with primary anti-phospho-IRβ antibody and Alexa Fluor 680-conjugated secondary antibody, and then the

fluorescent signals were visualized. Scale bar = 20 µm.

190x142mm (300 x 300 DPI)


Figure 4―GW501516-activated PPARδ extends the duration of insulin-stimulated IRβ phosphorylation. A: HepG2 cells were treated with 100 nM GW501516 or DMSO for 30 min, and then treated with or without 100

nM insulin for a further 30 min. Whole-cell lysates were prepared and subjected to immunoblot analysis.

HepG2 cells co-transfected with HA-tagged plasmids expressing hTCPTP45 or hTCPTP45350/378-A3 (B), or hTCPTP45 or hTCPTP48 (C), for 48 h were treated with 100 nM GW501516 or DMSO for 30 min, and then exposed to insulin for a further 30 min. Whole-cell lysates were prepared and subjected to immunoblot analysis. D and E: HepG2 cells (D) or primary hepatocytes (E) were transfected with or without control siRNA or siRNA targeting hTCPTP for 48 h, and then treated with 100 nM GW501516 or DMSO. After

incubation for 30 min, the cells were treated with or without 100 nM insulin for a further 30 min. Whole-cell lysates were analyzed by immunoblotting. F: HepG2 Cells were treated with 100 nM GW501516 or DMSO for

30 min, and then exposed to 100 nM insulin for the indicated periods of time. G: HepG2 cells transfected with Flag-hPPARδ for 48 h were treated with 100 nM GW501516 or DMSO for 30 min, and then exposed to

100 nM insulin for the indicated periods of time. H: Immunoblot analyses of (F) and (G) were quantified and represented with DMSO (black circles), GW501516 (white circles), Flag-hPPARδ overexpression + DMSO


(black triangles) and Flag-hPPARδ overexpression + GW501516 (white triangles). Data are expressed as mean ± SE (n = 3). *p < 0.01 vs DMSO-treated group. #p < 0.01 vs DMSO-treated Flag-hPPARδ group. I-K: HepG2 cells stably expressing control shRNA (I) or shRNA targeting PPARδ (J) were treated with 100 nM GW501516 or DMSO for 30 min, and then exposed to 100 nM insulin for the indicated periods of time. The

immunoblots were then quantified and represented with control shRNA + DMSO (black circles), control shRNA + GW501516 (white circles), hPPARδ shRNA + DMSO (black triangles) and hPPARδ shRNA +

GW501516 (white triangles) (K). Results are expressed as mean ± SE (n = 3). *p < 0.01 vs DMSO-treated control shRNA group. L-N: HepG2 cells stably expressing control shRNA (L) or shRNA targeting PPARδ (M) were transfected with either pcDNA-Flag or pcDNA-Flag-hPPARδ for 48 h. The cells were treated with 100

nM GW501516 or DMSO for 30 min, and then exposed to 100 nM insulin for a further 30 min. Immunoblots were then quantified and represented with control shRNA (red bars) or hPPARδ shRNA (blue bars) (N).

Results are expressed as mean ± SE (n = 3). **, ##p < 0.05.

271x320mm (300 x 300 DPI)


Figure 5―The GW501516-stimulated interaction of PPARδ with TCPTP45 directly affects the insulin receptor. A: HepG2 cells stably expressing PPARδ shRNA were co-transfected with pcDNA-HA, pcDNA-HA-hTCPTP45, pcDNA-Flag, and/or pcDNA-Flag-hPPARδ for 48 h. The cells were treated with 100 nM GW501516 or DMSO

for 30 min, and then exposed to 100 nM insulin for a further 30 min. Whole-cell lysates were analyzed by immunoblotting. B and C: HepG2 cells co-transfected with pcDNA-Flag-hPPARδ and pcDNA-HA-hTCPTP45

(B) or pcDNA-HA-hTCPTP45350/378-A3 (C) for 48 h were treated with 100 nM GW501516 or DMSO for 30 min, and then exposed to 100 nM insulin for a further 30 min. Cell lysates were then immunoprecipitated with anti-Flag antibody and analyzed by immunoblotting. D: HepG2 cells stably expressing PPARδ shRNA were treated with 100 nM GW501516 or DMSO for 30 min, and then exposed to 100 nM insulin for the

indicated periods of time. Whole-cell lysates were analyzed by immunoblotting. E: HepG2 cells co-transfected with pcDNA-Flag-hPPARδ and pcDNA-HA-hTCPTP45 or pcDNA-HA-hTCPTP45350/378-A3 for 48 h were treated with 100 nM GW501516 or DMSO for 30 min, and then exposed to 100 nM insulin for a further

6 h. Whole-cell lysates were immunoprecipitated and analyzed by immunoblotting. F: HepG2 cells stably expressing shRNA against control or PPARδ were treated with 100 nM GW501516 or DMSO for 30 min.

Following exposure to 100 nM insulin for 8 h, the quantity of glucose present in the glucose production medium was determined and represented with control shRNA (red bars) or hPPARδ shRNA (blue bars).

Results are expressed as mean ± SE (n = 3). **p < 0.05.

135x101mm (300 x 300 DPI)


Figure 6―GW501516-activated PPARδ modulates downstream insulin sensitivity and resistance through its interaction with TCPTP45. Differentiated C2C12 myotubes (A and C) or 3T3-L1 adipocytes (B and D) were treated with 100 nM GW501516 or DMSO for 30 min and then exposed to 100 nM insulin for the indicated

periods of time. The cells were subjected to immunoblot (A and B) or fluorescence image analysis (B and D). For Glut4 translocation assay, the fixed cells were sequentially reacted with primary anti-Glut4 antibody and

Alexa Fluor 488-conjugated secondary antibody, and then the fluorescent signals were visualized. Nuclei were stained with PI. Scale bars = 20 µm. E: HepG2 cells treated with or without 20 ng/ml IL-6 for 90 min

were incubated with 100 nM GW501516 or DMSO for 30 min, and then exposed to 100 nM insulin for a further 30 min. The cells were lysed and analyzed by immunoblotting. F: HepG2 cells stably expressing

control shRNA or shRNA targeting PPARδ were treated with or without 20 ng/ml IL-6 for 90 min. The cells were then incubated with 100 nM GW501516 or DMSO for a further 30 min, and then exposed to 100 nM

insulin for an additional 30 min. Whole-cell lysates were then analyzed by immunoblotting. G and H: HepG2 cells co-transfected with pcDNA-Flag-hPPARδ and pcDNA-HA-hTCPTP45 or pcDNA-HA-hTCPTP45350/378A3 (G), or primary hepatocytes (H) were treated with or without 20 ng/ml IL-6 for 90 min. The cells were then

incubated with 100 nM GW501516 or DMSO for a further 30 min, and then exposed to 100 nM insulin for a final 30 min. Whole-cell lysates were immunoprecipitated and analyzed by immunoblotting. Two percent of

each whole-cell lysate was used as the input.

190x142mm (300 x 300 DPI)


Figure 7―Administration of GW501516 improves in vivo insulin sensitivity and glucose tolerance through its interaction with TCPTP45. Mice fed a ND or HFD for 10 weeks were fasted for 14 h, and treated with 10 mg/kg GW501516 or DMSO for 30 min. Following an intraperitoneal injection of 2 g/kg glucose or 0.5

units/kg insulin, glucose (A) or insulin (B) tolerance tests were performed. Blood glucose was then measured and represented with ND + DMSO (black circles), ND + GW501516 (white circles), HFD + DMSO (black triangles) and HFD + GW501516 (white triangles). Data are expressed as mean ± SE (n = 6). *p <

0.01 vs HFD + DMSO group. Mice fed a ND or HFD for 10 weeks were fasted for 14 h, and then treated with 10 mg/kg GW501516 or DMSO for 30 min. Following exposure to 0.5 units/kg insulin for 30 min, the mice were sacrificed by inhalation of CO2 gas, and then liver (C), skeletal muscle (D), and adipose tissue (E)

were removed. Tissue homogenates were prepared and immunoprecipitated with anti-TCPTP45 antibody. The total lysates (input) and immunoprecipitates were analyzed by immunoblotting. Two percent of each tissue lysate was used as the input. Mice were fed a ND or HFD for 10 weeks and then starved for 14 h.

They were then treated with 10 mg/kg GW501516 (red bars) or DMSO (blue bars) for 30 min and sacrificed. Circulating levels of insulin and glucose were determined by ELISA or spectrometric methods using sera

prepared from blood collected from the carotid artery. Data are expressed as mean ± SE (n = 6). *, #p < 0.01, ##p < 0.05.

135x101mm (300 x 300 DPI)


1

SUPPLEMENTARY DATA

Materials

GSK0660 and GW501516 were purchased from Enzo Life Sciences (Farmingdale, NY, USA)

and Tocris Bioscience (Bristol, UK), respectively. Insulin, puromycin, isopropyl β-D-1-

thiogalactopyranoside (IPTG), propidium iodide (PI), mouse anti-Flag monoclonal antibody,

rabbit anti-β-actin polyclonal antibody, and HRP-conjugated goat anti-rabbit IgG antibody

were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Recombinant human IL-6

was obtained from R&D Systems (Minneapolis, MN, USA). A monoclonal anti-HA antibody

and polyclonal antibodies specific for phospho-Akt (Ser4473), Akt, and TCPTP45 were

obtained from Cell Signaling (Beverly, MA, USA). Monoclonal antibodies specific for

PTP1B, TCPTP, phospho-STAT3 (Tyr705), STAT3, and a polyclonal rabbit anti-SOCS3

antibody were obtained from Abcam (Cambridge, MA, USA). Polyclonal antibodies specific

for phospho-IRS1 (Tyr612), IRS, and Glut4 were obtained from Millipore (Bedford, MA,

USA). A rabbit anti-phospho-IR (Tyr1162/1163) polyclonal antibody was obtained from

Invitrogen (San Diego, CA, USA). Polyclonal antibodies specific for PPARα, PPARδ, and

PPARγ, and monoclonal antibodies specific for α-tubulin, lamin B, and GST, were purchased

from Santa Cruz Biotechnology (Dallas, TX, USA). ND (4.3% fat, 67.3% carbohydrate, and

19.2% protein) were purchased from Altromin Spezialfutter GmbH & Co. (Lage, North

Rhine-Westphalia, Germany) and a HFD (24% fat, 41% carbohydrate, and 24% protein

(206.8 g/kg lard)) were purchased from Research Diets Inc. (New Brunswick, NJ, USA).

Cell culture and differentiation

The human hepatoblastoma-derived HepG2 and HEK293T cells, and mouse 3T3-L1

preadipocytes, were obtained from the Korean Cell Line Bank (Seoul, Korea), and mouse


2

C2C12 myoblast cells were a gift from Dr. Eun Jung Cho (School of Pharmacy,

Sungkyunkwan University, Suwon, Korea). All were maintained in Dulbecco’s modified

Eagle’s medium (DMEM). CHO-IR cells were kindly provided by Dr. Deok-Bae Park

(Department of Medicine, Jeju National University, Jeju, Korea) and cultured in Ham’s F-12

medium. The primary hepatocytes from mouse were isolated by collagenase perfusion

method as described previously (1) and then cultured in DMEM/F-12 medium supplemented

with 0.1 µM dexamethasone, 10 mM nicotinamide, 1% insulin-transferrin-selenium premix,

and growth factors. All cells were maintained in medium containing antibiotics and 10% fetal

bovine serum at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. For the

differentiation of C2C12 myoblasts, cells were grown to near 100% confluency in normal

DMEM, as described above, and then incubated in DMEM containing 2% horse serum for 3

days to induce differentiation into myotubes, as described previously (2). To differentiate

3T3-L1 preadipocytes, cells were cultured to near 100% confluence, then incubated in

DMEM containing MDI (250 nM dexamethasone, 500 µM 3-isobutyl-1-methylxanthine, and

200 nM insulin) solution for 10 days to induce differentiation into adipocytes, as described

previously (3). For further analysis, the myotubes, adipocytes, and HepG2 cells were

incubated in serum-free DMEM for 16 h, and then treated with the indicated reagents for the

indicated periods of time.

Plasmid construction and transfection

To express epitope-tagged proteins, Flag- or HA-tagged pcDNA3.1 vector was used

(Stratagene, La Jolla, CA, USA). First-strand synthesis of full-length cDNAs for human

PPARδ (GenBank accession no. NM_006238.4), mouse PPARδ (GenBank accession no.

NM_011145.3), mouse PPARα (GenBank accession no. NM_011144.6), mouse PPARγ


3

(GenBank accession no. NM_001127330.2), human TCPTP48 (GenBank accession no.

NM_002828.3), human TCPTP45 (GenBank accession no. NM_080422.2), and mouse

TCPTP45 (GenBank accession no. NM_008977.3) was performed using 1 µg of total RNA

and TOPscript RT DryMIX kit (Enzynomics, Seoul, Korea). Reverse transcription was

followed by 25 cycles of PCR amplification using the primers 5ʹ-

CTGAAGCTTATGGAGCAGCCACAGGAG-3ʹ and 5ʹ-

AGTTCTAGATTAGTACATGTCCTTGTA-3ʹ (human PPARδ), 5ʹ-

GCCAAGCTTATGGAACAGCCACAGGAG-3ʹ and 5ʹ-

GCCGATATCTTAGTACATGTCCTTGTAG-3ʹ (mouse PPARδ), 5ʹ-

GCCAAGCTTATGGTGGACACAGAGAG-3ʹ and 5ʹ-

GCCGATATCTCAGTACATGTCTCTGTAG-3ʹ (mouse PPARα), 5ʹ-

GCCAAGCTTATGGTGGACACAGAGAG-3ʹ and 5ʹ-

GCCGATATCTCAGTACATGTCTCTGTAG-3ʹ (mouse PPARγ), 5ʹ-

CTGGGATCCATGCCCACCACCATCGAG-3ʹ and 5ʹ-

AGTCTCGAGTTATAGGGCATTTTGCTG-3ʹ (human TCPTP48), 5ʹ-

CTGGGATCCATGCCCACCACCATCGAG-3ʹ and 5ʹ-

AGTGATATCTTAGGTGTCTGTCAATCT-3ʹ (human TCPTP45), and 5ʹ-

CTGGGATCCATGTCGGCAACCATCGAG-3ʹ and 5ʹ-

AGTCTCGAGTTAGGTGTCTGTCAATCT-3ʹ (mouse TCPTP45). Each PCR product was

digested with appropriate restriction enzymes, and ligated into the similarly digested

pcDNA3.1/Flag or pcDNA3.1/HA vector to yield the expression vectors pcDNA3.1-Flag-

hPPARδ, pcDNA3.1-Flag-mPPARδ, pcDNA3.1-Flag-mPPARα, pcDNA3.1-Flag-mPPARγ,

pcDNA3.1-HA-hTCPTP48, pcDNA3.1-HA-hTCPTP45, and pcDNA3.1-HA-mTCPTP45,

respectively. The pMT2-hTCPTP45D182A

plasmid was a gift from Dr. Nicholas K. Tonks


4

(Cold Spring Harbor Laboratory, New York, USA). For GST-fusion proteins, full-length

hTCPTP45 cDNA was cloned into the BamHI and XhoI sites of the pGEX4T-1 vector (GE

Healthcare Life Sciences, PA, USA) to yield the GST-fusion vector pGEX4T-1-hTCPTP45.

Deletion mutants of GST-hTCPTP45 were constructed by PCR amplification of fragments

from pGEX4T-1-hTCPTP45. These fragments were digested with BamHI/XhoI and ligated

into the similarly digested pGEX4T-1 vector, generating GST-hTCPTP45 (D1) and GST-

hTCPTP45 (D2). Site-directed mutants of GST-hTCPTP45, GST-hTCPTP45350-A3

(R350A,

K351A, and R351A), GST-hTCPTP45378-A3

(K378A, R379A, and K380A), and GST-

hTCPTP45350/378-A3

(R350A, K351A, R351A, K378A, R379A, and K380A) were created

using a QuikChange Site-Directed Mutagenesis Kit (Stratagene) and pGEX4T-1-hTCPTP45

plasmid. HA-tagged hTCPTP45350/378-A3

was generated by PCR amplification of GST-

hTCPTP45350/378-A3

using the primers 5ʹ- CTTAAGCTTATGAGCGATAACGATGAC-3ʹ and

5ʹ-CCATCTAGATCAGCTCTCGCTTTCCCCTT-3ʹ. The PCR product was digested with

HindIII and XbaI, and ligated into the similarly digested pcDNA3.1/HA vector to yield the

expression vector pcDNA3.1-HA-hTCPTP45350/378-A3

. For the localization assay, GFP-

hPPARδ, DsRed-hTCPTP45D182A

, and DsRed-hTCPTP48D182A

were generated using pEGFP-

C1 and pDsRed-Express-C1 (Clontech Laboratories, Inc., CA, USA). All recombinant

plasmids were sequenced and verified. HepG2 and HEK293T cells were transfected with the

indicated plasmids using Genefectin (Genetrone Biotech, Gwangmyeong, Korea) for 6 h, and

the existing medium was then replaced with fresh medium. After incubation for an additional

48 h, the cells were stimulated with the indicated reagents for the specified period of time.

Bacterial expression of GST-fusion proteins and GST pull-down assay

The binding of PPARδ to TCPTP45 was assessed using bacterially expressed GST-fusion


5

proteins essentially as previously described (4). Briefly, mouse full-length GST-hTCPTP45

(FL), GST-hTCPTP45 (D1), GST-hTCPTP45 (D2), GST-hTCPTP45350-A3

, GST-

hTCPTP45378-A3

, and GST-hTCPTP45350/378-A3

cloned into the pGEX4T-1 vector were

transformed into BL21 competent cells. Clones were cultured in LB medium containing 50

µg/ml ampicillin, and the expression of GST or GST-fused hTCPTP45 was initiated with 0.5

mM IPTG at an OD600 of approximately 0.6 for 18 h at 18 °C. The bacterial pellets were

collected by centrifugation at 10,000 × g for 15 min and then resuspended in lysis buffer (30

mM Tris-Cl, 1% NP-40, protease inhibitors, 1 mM DTT, 0.1 mM NaCl, pH 7.5). The

bacterial lysates were sonicated for 3 × 1 min, and the soluble fraction was obtained by

centrifugation at 12,000 rpm for 30 min. Because the GST-fusion proteins were largely in the

soluble fraction, the supernatants were mixed with glutathione-Sepharose 4B beads (GE

Healthcare Life Sciences) for 4 h to immobilize the GST-fusion proteins. To identify binding

of PPARδ to TCPTP45, the immobilized GST-fusion proteins were mixed with cell lysates

overexpressing PPARδ in the presence or absence of GW501516 overnight at 4 °C.

Following extensive washing with phosphate-buffered saline (PBS), the interaction between

PPARδ and TCPTP45 was evaluated by immunoblotting.

Co-immunoprecipitation and immunoblot analysis

Cell and tissue lysates were prepared using PRO-PREP Protein Extraction Solution (iNtRON

Biotechnology, Seoul, Korea) and then pre-cleared with protein G Sepharose™ 4 Fast Flow

(GE Healthcare Life Sciences, Buckinghamshire, UK). Pre-cleared lysates were mixed with

the specified antibodies (1 µg) or relevant IgG overnight at 4 °C, and then mixed with protein

G Sepharose for 4 h. After washing with PBS, proteins were separated from the Sepharose


6

beads by boiling in 2×SDS gel-loading buffer and resolved on 10% SDS-polyacrylamide gels.

The immunoprecipitates and total lysates (input) were subjected to immunoblot analysis with

the specified antibodies, and immunoreactive bands were detected using West-ZOL Plus

(iNtRON Biotechnology), as previously described (4). Two percent of each whole-cell lysate

was used as the input.

Creation of HepG2 cells stably expressing shRNA targeting PPARδδδδ

PPARδ-silenced HepG2 cells were created by infection with lentiviral particles expressing

PPARδ (TRCN0000350974, MISSION Lentiviral Transduction Particles, Sigma-Aldrich) or

a control containing a puromycin resistance gene (pLKO.1-puro Non-Target shRNA Control

Transduction Particles, Sigma-Aldrich). The viral particles were used to infect HepG2 cells

according to the manufacturer’s instructions, and positive cells were selected using 2 µg/ml

puromycin. The efficacy of PPARδ silencing was confirmed by immunoblot analysis.

Gene silencing with siRNA

Gene silencing with specific siRNAs was performed essentially as previously described (5).

Briefly, HepG2 cells were transfected with transfection-ready control siRNA (Ambion,

Austin, TX, USA), human PPARδ-targeting siRNA (Ambion), and human TCPTP-targeting

siRNA designed against the sequences 5ʹ-GGUCCACUUCCUAACACAUGCdTdT-3ʹ and

5ʹ-GCAUGUGUUAGGAAGUGGACCdTdT-3ʹ of the PTPN2 mRNA, and human PTP1B-

targeting siRNA designed against the sequences 5ʹ-UAGGUACAGAGACGUCAGUdTdT-3ʹ

and 5ʹ-ACUGACGUCUCUGUACCUAdTdT-3ʹ of the PTPN1 mRNA, in serum-free DMEM.

The siRNAs against TCPTP and PTP-1B were synthesized by Bioneer (Daejeon, Korea).


7

Following incubation for 6 h, the medium was replaced with fresh medium containing 10%

FBS and the cells were incubated for an additional 24 h. The cells were then serum-starved

for 18 h and treated with the specified reagents for the indicated period of time, after which

the effects of gene silencing were assessed.

Fluorescence confocal laser microscopy

Fluorescence images were analyzed as described previously (4). Briefly, 1 × 104 cells were

seeded on cover-glasses in 35 mm dishes (SPL Life Sciences, Seoul, Korea), and then

transfected with GFP-hPPARδ and DsRed-hTCPTP45D182A

or DsRed-hTCPTP48D182A

using

Genefectin (Genetrone Biotech). Forty-eight hours after transfection, cells were treated with

insulin, GW501516, and/or GSK0660 for the indicated periods of time. The cells were then

incubated with 2 µg/ml DAPI (4′,6-diamidino-2-phenylindole) solution for 10 min at room

temperature. Following staining, the cover-glasses were mounted and examined using an

Olympus FV-1000 confocal laser fluorescence microscope (Olympus, Tokyo, Japan). The line

intensity profiles were obtained using Image J 1.5 (imagej.nih.gov/ij/) software.

Glut4 translocation assay

C2C12 myotubes and 3T3-L1 adipocytes seeded on 35 mm cover-glasses in dishes (SPL Life

Sciences) were incubated in serum-free DMEM for 16 h and then stimulated with 100 nM

insulin in the presence or absence of GW501516. After treatment for 30 min or 6 h, the cells

were fixed using neutral buffered 4% formaldehyde solution for 7 min. After

permeabilization with PBS containing 0.1% Tween-20 for 3 min, the fixed cells were

incubated overnight at 4 °C with 1:200 anti-Glut4 antibody. Secondary goat anti-rabbit IgG

conjugated to Alexa Fluor 488 (Sigma) was applied for 2 h at room temperature, and then the


8

cells were incubated for 10 min in PI solution. The subcellular localization of Glut4 was

evaluated in single cells using an Olympus FV-1000 confocal laser fluorescence microscope.

Hepatocyte glucose production assay

The glucose content of media was measured according to a previously described method (1).

Briefly, HepG2 cells seeded on a 96-well plate were incubated in serum-free DMEM for 16 h

and then the medium was replaced with glucose production medium (glucose- and phenol

red-free DMEM supplemented with 20 mM sodium lactate and 2 mM sodium pyruvate).

HepG2 cells were treated with 100 nM GW501516 or vehicle for 30 min, and then exposed

to 100 nM insulin for 8 h. The glucose concentration in the conditioned medium was

measured using a QuantiChrom Glucose assay kit (Bioassay Systems, CA, USA) and

normalized to total protein content.

Animal study

Male ICR mice (4-week-old, 15–20 g) were purchased from Orient Bio (Seongnam, Korea)

and maintained in pathogen-free environmental conditions on a 12 h light-dark cycle at 22 ±

2 °C. All procedures were approved by the Institutional Animal Care and Use Committee of

Konkuk University (approval number: KU16144). After 1 week of acclimation, the mice

were divided into two groups, and were fed either a ND (Altromin Spezialfutter GmbH & Co.,

Lage, Germany) or a HFD (Research Diets, Inc. NJ, USA) for 10 weeks, during which water

and diet were available ad libitum. The ND contained 4.3% fat, 67.3% carbohydrate, and 19.2%

protein, whereas the HFD was composed of 24% fat, 41% carbohydrate, and 24% protein

(206.8 g/kg lard). Body mass was measured once a week.


9

Serum analysis

Serum levels of insulin, glucose, and IL-6 were analyzed in circulating blood samples

obtained from ND or HFD-fed mice that had been starved for 14 h and treated with 10 mg/kg

GW501516 or DMSO for 30 min. Blood was collected, allowed to clot for 2 h at room

temperature, and centrifuged for 20 min at 4,000 rpm, as described previously (4). Serum

glucose was determined using an OneTouch automatic glucose monitor (LifeScan, Inc.).

Serum levels of insulin and IL-6 were measured using a Ultra Sensitive Mouse Insulin ELISA

Kit (Morinaga Institute of Biological Science, Inc., Yokohama, Japan) and a Mouse IL-6

Platinum ELISA Kit (eBioscience, San Diego, CA, USA), respectively.


10

Supplementary Figure 1―Ligand-dependent interaction of PPARδ with TCPTP45. A:

HepG2 cells were transfected with control shRNA or shRNA targeting PPARδ, and the stable

transfectants were selected in the presence of 2 µg/ml hygromycin. The transfectants were

lysed and immunoblotted to determine the expression levels of PPARδ and β-actin. B:

HEK293T cells co-transfected with Flag-hPPARδ and/or HA-hTCPTP45 for 48 h were

treated with 100 nM GW501516 or DMSO for the indicated periods of time, and then whole-

cell lysates were prepared and immunoprecipitated with anti-Flag antibody. Immunoblot

analysis was performed using the immunoprecipitates and total lysates (input). C: HEK293T

cells co-transfected with pcDNA-Flag, pcDNA-Flag-mPPARα, pcDNA-Flag-mPPARδ, or

pcDNA-Flag-mPPARγ in the presence of pcDNA-HA-mTCPTP45 for 48 h were treated with

10 µM WY-14643 (PPARα activator), 100 nM GW501516 (PPARδ activator), or 10 µM

rosiglitazone (PPARγ activator) for 45 min, and then whole-cell lysates were prepared and

immunoprecipitated with anti-Flag antibody. Immunoblot analysis was performed using the

immunoprecipitates and total lysates (input). Two percent of each whole-cell lysate was used

as the input.


11

Supplementary Figure 2―Interaction of PPARδ with TCPTP in subcellular fractions. A:

HepG2 cells were treated with 100 nM GW501516 or DMSO for 45 min, and then whole-cell

lysates were prepared and immunoprecipitated with IgG or anti-PPARδ antibody. The

immunoprecipitates and total lysates (input) were subjected to immunoblot analysis. B and C:

HepG2 cells were treated with 100 nM GW501516 or DMSO for 45 min, and then separated

into nuclear (B) and cytoplasmic (C) fractions. Each subcellular fraction was

immunoprecipitated with anti-PPARδ antibody. D: Schematic illustration of the C-terminal

bipartite NLS region of TCPTP45 replaced with canonical SV40 large T antigen NLS

(PKKKRKV) by site-directed mutation to yield mutant TCPTP45350/378-SV40

. E: HepG2 cells

were transfected with HA-hTCPTP45350/378-SV40

for 48 h and then separated into nuclear (N)

and cytoplasmic (C) fractions. Each fraction was analyzed by immunoblot. F: HepG2 cells

co-transfected with Flag-hPPARδ and HA-hTCPTP45350/378-SV40

for 48 h were treated with

100 nM GW501516 or DMSO for 30 min. Following exposure to 100 nM insulin for 30 min,


12

the cells were lysed and immunoprecipitated with anti-Flag antibody. The

immunoprecipitates and total lysates (input) were subjected to immunoblot analysis. Two

percent of each whole-cell lysate was used as the input.


13

Supplementary Figure 3―Localization of PPARδ and TCPTP45 or TCPTP48 complexes. A

and B: CHO-IR cells co-transfected with GFP-hPPARδ and DsRed-hTCPTP45D182A

(A) or

DsRed-hTCPTP48D182A

(B) for 48 h were treated with 100 nM GW501516 or DMSO for 30

min, and then exposed to 100 nM insulin or vehicle. After a further incubation for 30 min, the

fluorescence of each fusion protein was visualized using confocal laser fluorescence

microscopy. The co-localization of PPARδ and TCPTP45 or TCPTP48 is indicated by the

presence of white in the merged image. Representative fluorescence intensity profiles across

the cell were obtained from the merged images through the white arrows. Scale bar = 20 µm.


14

Supplementary Figure 4―Differential effects of PPAR ligands on the insulin-stimulated

phosphorylation of IRβ. A: HepG2 cells transfected with or without control siRNA or siRNA

targeting PTP1B for 48 h were treated with 100 nM GW501516 or DMSO for 30 min, and

then exposed to 100 nM insulin or vehicle for a further 30 min. Whole-cell lysates were

analyzed by immunoblotting to detect the indicated proteins using specific antibodies. B:

HepG2 cells were transfected with pcDNA-Flag or pcDNA-Flag-hPPARδ for 48 h and then

lysed and immunoblotted with anti-PPARδ and anti-β-actin antibodies to detect PPARδ and

β-actin, respectively. C and D: HepG2 cells transfected with pcDNA-Flag (C) or pcDNA-

Flag-hPPARδ (D) for 48 h were treated with 100 nM GW501516 or DMSO for 30 min and

then exposed to 100 nM insulin for the indicated periods of time. Immunoblot analysis was

performed using whole-cell lysates to detect the indicated proteins using specific antibodies.

E and F: HepG2 cells treated with 10 µM WY-14643 (E) or 10 µM rosiglitazone (F), specific

ligands for PPARα and PPARγ, for 30 min were exposed to 100 nM insulin or vehicle for the

indicated time periods. The cells were then lysed and analyzed by immunoblotting.


15

Supplementary Figure 5―Involvement of TCPTP45 in the PPARδ-mediated potentiation of

insulin signaling. A: HepG2 cells stably expressing control shRNA were transfected with

pcDNA-HA or pcDNA-HA-hTCPTP45 for 48 h. The cells were then treated with 100 nM

GW501516 or DMSO for 30 min, and exposed to 100 nM insulin for a further 30 min.

Whole-cell lysates were prepared and subjected to immunoblot analysis using specific

antibodies. B: HepG2 cells stably expressing control shRNA were treated with 100 nM

GW501516 or DMSO for 30 min, and then exposed to 100 nM insulin for the indicated

periods of time. Whole-cell lysates were prepared and subjected to immunoblot analysis.


16

Supplementary Figure 6―PPARδ-mediated modulation of insulin signaling. A and B:

C2C12 myotubes (A) or 3T3-L1 adipocytes (B) were treated with 100 nM GW501516 or

DMSO for 30 min. Following exposure to 100 nM insulin for 30 min, the cells were lysed

and sequentially analyzed by immunoprecipitation and immunoblotting. C: C2C12 myotubes

co-transfected with Flag-hPPARδ and HA-hTCPTP45 or HA-hTCPTP45350/378-A3

for 48 h

were treated with 100 nM GW501516 or DMSO for 30 min, and then exposed to 100 nM

insulin for a further 30 min. Whole-cell lysates were prepared and analyzed by


17

immunoprecipitation and immunoblotting. Two percent of each whole-cell lysate was used as

the input. D: HepG2 cells transfected with siRNA were treated with or without 20 ng/ml IL-6.

Following treatment for 90 min, the cells were incubated with 100 nM GW501516 or DMSO

for 30 min, and then exposed to 100 nM insulin for a further 30 min. The cells were lysed and

subjected to immunoblot analysis.


18

Supplementary Figure 7―In vivo effects of GW501516 in mice fed a normal or high-fat

diet. A: Time-dependent changes in body mass of mice fed a ND (black circles) or HFD

(white circles) for 10 weeks. Data are expressed as mean ± SE (n = 6). *p < 0.01 and

**p <

0.05 vs ND group. B: Mice fed a ND or HFD for 10 weeks were starved for 14 h and then

treated with 10 mg/kg GW501516 (red bars) or DMSO (blue bars) for 30 min. Circulating

levels of IL-6 were measured by ELISA using sera prepared from blood collected from the

carotid artery. Data are expressed as mean ± SE (n = 4). **p < 0.05.


19

References

1. Choi D, Oh KJ, Han HS, et al. Protein arginine methyltransferase 1 regulates hepatic

glucose production in a FoxO1-dependent manner. Hepatology 2012;56:1546-1556.

2. Song, TY, Yang, JH, Park, JY, et al. The role of histone chaperones in osteoblastic

differentiation of C2C12 myoblasts. Biochem Biophys Res Commun. 2012;423:726-732.

3. Zebisch, K, Voigt V, Wabitsch M, et al. M. Protocol for effective differentiation of 3T3-L1

cells to adipocytes. Anal Biochem. 2012;425:88-90.

4. Hwang JS, Choi HS, Ham SA, et al. Deacetylation-mediated interaction of SIRT1-HMGB1

improves survival in a mouse model of endotoxemia. Sci Rep 2015;5:15971.

5. Yoo T, Ham SA, Hwang JS, et al. Peroxisome proliferator-activated receptor δ inhibits

Porphyromonas gingivalis lipopolysaccharide-induced activation of matrix

metalloproteinase-2 by downregulating NADPH oxidase 4 in human gingival fibroblasts.

Mol Oral Microbiol 2016;31:398-409.


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