Role of Plasminogen Activator Inhibitor-1 in Glucocorticoid ... - …diabetes.diabetesjournals.org/content/diabetes/early/2014/12/24/db... · 0.05 were considered significant. All

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Role of Plasminogen Activator Inhibitor-1 in Glucocorticoid-Induced Diabetes and

Osteopenia in Mice

Yukinori Tamura,1 Naoyuki Kawao,

1 Masato Yano,

1 Kiyotaka Okada,

1 Katsumi

Okumoto,2 Yasutaka Chiba,

3 Osamu Matsuo,

4 and Hiroshi Kaji

1*

1. Department of Physiology and Regenerative Medicine, Kinki University Faculty of

Medicine, Osakasayama, Japan

2. Life Science Research Institute, Kinki University, Osaka, Japan

3. Clinical Research Center, Kinki University Hospital, Osakasayama, Japan

4. Kinki University Faculty of Medicine, Osakasayama, Japan

*To whom correspondence should be addressed:

Hiroshi Kaji, M.D., Ph.D., Department of Physiology and Regenerative Medicine,

Kinki University Faculty of Medicine, 377-2 Ohnohigashi, Osakasayama, Osaka

589-8511, Japan

Tel.: +81 72-366-0221 (ext. 3165) Fax: +81 72-366-2853

Page 1 of 50 Diabetes

Diabetes Publish Ahead of Print, published online December 31, 2014

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Short Running Title: PAI-1 AND GLUCOCORTICOID-INDUCED ADVERSE

EFFECT

Words count: 3993 words

Page 2 of 50Diabetes

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ABSTRACT

Long-term use of glucocorticoids (GCs) causes numerous adverse effects, including

glucose/lipid abnormalities, osteoporosis, and muscle wasting. The pathogenic

mechanism, however, is not completely understood. In this study, we used

PAI-1-deficient mice to explore the role of plasminogen activator inhibitor 1 (PAI-1) in

GC-induced glucose/lipid abnormalities, osteoporosis, and muscle wasting.

Corticosterone markedly increased the levels of circulating PAI-1 and the PAI-1 mRNA

level in the white adipose tissue of wild-type mice. PAI-1 deficiency significantly

reduced insulin resistance and glucose intolerance but not hyperlipidemia induced by

GC. An in vitro experiment revealed that active PAI-1 treatment inhibits insulin-induced

phosphorylation of Akt and glucose uptake in HepG2 hepatocytes. However, this was

not observed in 3T3-L1 adipocytes and C2C12 myotubes, indicating that PAI-1

suppressed insulin signaling in hepatocytes. PAI-1 deficiency attenuated the

GC-induced bone loss presumably via inhibition of apoptosis of osteoblasts. Moreover,

the PAI-1 deficiency also protected from GC-induced muscle loss. In conclusion, the

present study indicated that PAI-1 is involved in GC-induced glucose metabolism

abnormality, osteopenia, and muscle wasting in mice. PAI-1 may be a novel therapeutic

target to mitigate the adverse effects of GC.


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KEY WORDS: Glucocorticoid, Insulin Resistance, Osteoporosis, PAI-1, Muscle

Weakness


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INTRODUCTION

Glucocorticoids (GCs) have strong anti-inflammatory properties and are highly

effective in the treatment of allergies, inflammatory and autoimmune conditions, such as

rheumatoid arthritis, asthma, inflammatory bowel disease, and collagen diseases (1; 2).

Despite the high efficacy of GC treatment, its clinical use is limited due to adverse

effects, such as diabetes, hyperlipidemia, osteoporosis, muscle wasting, and

immunosuppression, which are depend on the administered dose and duration of GC

treatment (3-5).

GC-induced diabetes and osteoporosis are manifestations of adverse metabolic effects

because of the high incidence. A clinical study showed that more than 11% of GC users

develop diabetes mellitus within 3 years of GC therapy (6), and a larger number of

patients transition into a prediabetic state, such as insulin resistance and impaired

glucose tolerance. In muscles, GCs have been shown to suppress a number of steps in

the insulin signaling network (3; 7). GCs directly promote hepatic gluconeogenesis,

leading to hyperglycemia (7). Moreover, GCs promote proteolysis, lipolysis, free fatty

acid production, and fat accumulation in the liver that contributes to insulin resistance.

GC treatment is the most common cause of secondary osteoporosis (8). GCs affect

osteoblasts, osteoclasts, and osteocytes. Osteoblasts are generally considered the main


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skeletal target (4; 9). Several studies suggest that GCs suppress bone formation by

inhibiting differentiation, proliferation, and apoptosis of osteoblasts, leading to

osteoporosis (4; 9); however, the mechanism is not well understood. In addition, the

patients treated with GC frequently suffer from muscle wasting (10). GCs are also

important mediators of muscle wasting in many pathological conditions, such as sepsis,

cachexia, starvation, and metabolic acidosis (11).

Experimental evidence suggests that the above-mentioned effects of GCs are induced

in the cytoplasm by the direct action of GC binding to glucocorticoid receptors (GR) (4).

Nonetheless, the precise mechanisms are not fully understood, and the evidence of

systemic mediators, in the adverse effects of GCs, is lacking.

Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor that primarily

inhibits tissue-type and urokinase-type plasminogen activators; hence, it is an inhibitor

of fibrinolysis. PAI-1 is a well-known adipocytokine, being upregulated along with fat

accumulation (12). It has been suggested that elevated levels of circulating PAI-1 is a

risk factor in cardiovascular diseases (atherosclerosis), obesity, and diabetes (13-15).

Moreover, we recently demonstrated in female mice with streptozotocin-induced type 1

diabetes that PAI-1 is involved in bone loss (16). PAI-1 is upregulated in atrophic

skeletal muscle (17), and this change is associated with impaired muscle regeneration


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(18; 19). Several clinical studies showed that circulating PAI-1 concentration is elevated

in patients with Cushing syndrome or during corticosteroid treatment (20; 21).

Nonetheless, the role of PAI-1 in GC-induced glucose/lipid abnormalities, osteoporosis,

and muscle wasting is unknown. Therefore, we examined the effects of PAI-1

deficiency on GC-induced glucose/lipid and bone metabolism abnormalities as well as

muscle wasting in mice.

RESEARCH DESIGN AND METHODS

Animal experiments

Forty-four female and 24 male mice with a mixed C57BL/6J (81.25%) and 129/SvJ

(18.75%) genetic background were analyzed as described in Figure Legends. We

included 22 female and 12 male mice with PAI-1 gene deficiency (PAI-1 knockout

[KO]) and the corresponding wild-type (WT) control mice (22). These mice were kindly

provided by Professor D. Collen (University of Leuven, Leuven, Belgium).

Nine-week-old female WT and PAI-1 KO mice received a subcutaneous implant with

slow-release pellets containing either 1.5 mg of corticosterone or placebo (Innovative

Research of America, Sarasota, FL). These pellets were implanted on days 0, 7, 14, and

21 (23; 24). The numbers of female and male mice in each group were 11 and 6,


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respectively. The animals were maintained in metabolic cages in the 12-h light/12-h

dark cycle; they received food and water ad libitum. Four weeks after the first implant

of corticosterone or placebo pellets, insulin and glucose tolerance tests were performed.

Quantitative computed tomography (qCT) was used to measure bone mineral density

(BMD) in the tibia. Mice (placebo- and corticosterone-treated groups) were starved for

6 h before euthanization; blood and tissue samples were collected from the dead mice.

All experiments were performed according to the guidelines of the National Institute of

Health and as per the institutional rules put forth for the use and care of laboratory

animals at Kinki University.

Metabolic parameters

The plasma concentrations of insulin, total PAI-1, triglyceride (TG), total cholesterol

(T-Chol), osteocalcin (OCN), cross-linked C-telopeptide of type I collagen (CTX), and

TG content in liver and muscle were measured (25; 26). The plasma concentration of

uncarboxylated OCN (ucOCN) was measured using Mouse Undercarboxylated

Osteocalcin ELISA Kit (MyBiosource, San Diego, CA). Glucose and insulin tolerance

tests were performed as previously described (25).


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The qCT analysis

For the qCT analysis of body fat composition, BMD and muscle volume, the mice were

scanned using a LaTheta (LCT-200) experimental animal CT system (Hitachi-Aloka

Medical, Tokyo, Japan).

Histological analysis

The tibia, muscle, and liver tissues were fixed in 4% paraformaldehyde for 16 h at 4°C,

and the tibia was further fixed for 7 days in 80% ethanol. Muscle and liver tissues were

embedded in paraffin right away, and the tibia was embedded in paraffin after

dehydration with formic acid. A paraffin block was sliced into 4-µm sections. For

visualization of osteoclasts, the slices were stained using a tartrate-resistant acid

phosphatase (TRAP)/alkaline phosphatase (ALP) Staining Kit (Wako Pure Industries,

Osaka, Japan). Immunostaining with ALP was performed (27). Another type of staining,

terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), was

performed to identify apoptotic cells, using an in situ Cell Death Detection Kit,

Fluorescein (Roche Diagnostics, Tokyo, Japan). The numbers of osteoclasts,

TUNEL-positive and ALP-positive cells were counted in selected visual fields under a

microscope in a blinded evaluation (16; 27).


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Quantitative real-time PCR

Total RNA was extracted from the homogenized tissue samples and the cultured cells

using the RNeasy Mini Kit (Qiagen, Tokyo, Japan). Real-time PCR was performed

using StepOne Plus and the Fast SYBR green PCR Master Mix (Life Technologies,

Tokyo, Japan) as previously described (25). The primer sets are shown in

Supplementary Table 1. The mRNA levels in tissues of the mice and in the cultured

cells were normalized to β-actin and glyceraldehyde 3-phosphate dehydrogenase

(GAPDH) mRNA, respectively.

Cell culture

3T3-L1 cells and C2C12 myoblasts were induced to differentiate into adipocytes and

myotubes, respectively (28; 29). The differentiated C2C12, 3T3-L1 and HepG2 cells

were preincubated with human active PAI-1 (Molecular Innovations, Novi, MI) and

dexamethasone (Sigma Aldrich Japan, Tokyo, Japan) in the serum-free Dulbecco’s

modified Eagle’s medium (DMEM) for 24 h. The cells were incubated with 100 nM

insulin for 15 min. Protein levels of phosphorylated Akt (Ser473

) and total Akt were

measured by western blotting (30). The expressions of gluconeogenesis-related genes in


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hepatocytes were measured; HepG2 cells were incubated with 20 nM human active

PAI-1 and 100 nM dexamethasone for 24 h in the presence of as well as without 1 nM

insulin. Insulin-stimulated 2-deoxyglucose (2DG) uptake in HepG2, C2C12, and

3T3-L1 cells was assessed using 2-Deoxyglucose (2DG) Uptake Measurement Kit

(Cosmo Bio, Tokyo, Japan).

Cell proliferation and apoptosis in primary osteoblasts and MC3T3E1 cells

Primary osteoblastic cells were obtained from female WT and PAI-1 KO mouse calvaria

(27). Cell proliferation, apoptosis, and cell death rates of primary osteoblasts and

MC3T3E1 cells were analyzed using the bromodeoxyuridine (BrdU) Cell Proliferation

Assay Kit (Exalpha Biologicals, Shirley, MA), in situ Cell Death Detection Kit,

Fluorescein, and trypan blue staining, respectively.

Statistical analysis

All data were expressed as mean ± SEM. Two-way ANOVA was used to compare the

effects of the placebo (“Cont”) and corticosterone pellet (“CS”) on both the mice

genotypes (PAI-1 WT and PAI-1 KO). Specifically, 2-way repeated-measures of

ANOVA was used for repeated measured. When significant differences were observed,


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individual means were compared using Tukey’s post hoc test. For the other simple

comparisons between 2 groups, the unpaired t test was used. Statistical values at p <

0.05 were considered significant. All calculations were performed in the StatView

software, version 5.0 (SAS Institute).

RESULTS

Effects of GC on adiposity and levels of circulating PAI-1

Corticosterone treatment increased body weight in female and male WT mice,

compared to placebo-treated WT mice (Figures 1A, B). The qCT analysis showed that

the percentage (%) of fat mass, visceral fat, and subcutaneous fat mass were markedly

increased by corticosterone in both sexes of WT mice, compared to the corresponding

control WT mice (Figures 1C–F). The PAI-1 deficiency did not affect the body weight

gain, % of fat mass, visceral fat, and subcutaneous fat mass either in placebo- or

corticosterone-treated female and male mice (Figures 1A–F). The levels of plasma

PAI-1 were markedly higher in both sexes of corticosterone-treated WT mice, compared

to the corresponding control WT mice (Figures 1G, H). The levels of PAI-1 mRNA

were markedly and significantly elevated in adipose and muscle tissues but significantly

decreased in the liver and spleen of corticosterone-treated WT mice compared to


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placebo-treated WT mice. There were no differences in the levels of PAI-1 mRNA in

tibia, heart, lung, and kidney between the placebo- and corticosterone-treated WT mice

(Figure 1I).

Effects of the PAI-1 deficiency in GC-induced abnormal glucose and lipid

metabolism

The PAI-1 deficiency significantly suppressed the fasting levels of blood glucose and

the fasting plasma levels of insulin that were elevated by corticosterone in both sexes of

mice (Figures 2A–D).

The decrease in blood glucose levels after i.p. insulin injection was attenuated in

corticosterone-treated WT mice; the PAI-1 deficiency significantly reversed the insulin

intolerance of corticosterone-treated WT mice (Figures 2E, F), although the reversion of

blood glucose levels by the PAI-1 deficiency was not significant 90 and 120 minutes

after the insulin injection in male mice. The levels of blood glucose after i.p. glucose

injection were markedly elevated in corticosterone-treated WT mice compared to

placebo-treated WT mice (Figures 2G, H). The PAI-1 deficiency significantly

suppressed the elevation of blood glucose levels in corticosterone-treated mice 60, 90,

and 120 minutes after the glucose injection (Figures 2G, H). Moreover, the PAI-1


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deficiency significantly suppressed the levels of plasma insulin in the mice 30 min after

the glucose injection (Figures 2I, J).

Active PAI-1 significantly suppressed Akt phosphorylation and glucose uptake

induced by insulin in HepG2 cells (hepatocytes), although active PAI-1 did not affect

them in differentiated C2C12 cells (myotubes) and differentiated 3T3-L1 cells

(adipocytes; Figures 3A–F). On the other hand, dexamethasone suppressed Akt

phosphorylation and glucose uptake induced by insulin in HepG2, differentiated C2C12,

and differentiated 3T3-L1 cells (Figures 3A–F), whereas the effects of both

dexamethasone and PAI-1 treatment were not additive in HepG2 cells (Figures 3A, D).

Moreover, active PAI-1 and dexamethasone did not affect the mRNA levels of key

enzymes of gluconeogenesis such as glucose-6-phosphatase (G6Pase) and PEPCK in

the presence of insulin in HepG2 cells, although insulin suppressed them in the absence

of PAI-1 and dexamethasone (Figures 3G, H).

Corticosterone markedly increased the levels of plasma TG and T-Chol in female

and male WT mice compared to placebo-treated WT mice, whereas the PAI-1

deficiency did not affect the levels of these plasma lipids in corticosterone-treated mice

(Figures 4A–D). The PAI-1 deficiency did not seem to affect corticosterone-induced

lipid accumulation in the adipose tissue and liver of the mice in histological analysis


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(Supplementary Figures 1A, B). The PAI-1 deficiency did not affect hepatic and

muscular TG contents that were elevated by corticosterone (Supplementary Figures 1D,

E).

Effects of the PAI-1 deficiency on GC-induced bone loss

The qCT analysis revealed that corticosterone treatment significantly reduced BMD,

bone volume fraction (BV/TV) and trabecular area of the tibia in female and male WT

mice, compared to placebo-treated WT mice (Figures 5A–H, O, and P), although it did

not affect cortical thickness, cortical area, and total cross-sectional area in the tibia of

WT mice (Figures 5I–N). In contrast, the PAI-1 deficiency blunted the

corticosterone-induced decrease in BMD, BV/TV, and trabecular area in both sexes of

mice (Figures 5A–H, O, and P).

Effects of the PAI-1 deficiency in GC-induced abnormal bone metabolism

Corticosterone treatment markedly suppressed the expression levels of osteogenic genes,

such as Runx2, Osterix, Alp, Ocn, and type 1 collagen (Col1) in tibia, the levels of

plasma OCN and ucOCN in WT mice, compared to the placebo-treated WT mice

(Figures 6A-C). The PAI-1 deficiency did not affect these osteogenic markers, when


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they were downregulated by corticosterone in mice (Figures 6A–C). In contrast,

immunohistochemical analysis revealed that the PAI-1 deficiency attenuated the

corticosterone-induced decrease in the number of ALP-positive osteoblastic cells on the

bone surface of tibia in mice (Figure 6D). When compared to the tibia of

placebo-treated WT mice, corticosterone suppressed the levels of receptor activator of

nuclear factor-кB ligand (RANKL) mRNA, osteoprotegerin (OPG) mRNA, the

RANKL/OPG ratio, and the number of TRAP-positive osteoclasts in the tibia of WT

mice (Figure 6E). In contrast, corticosterone did not affect the levels of plasma CTX, a

marker for bone resorption, in WT mice (Figure 6E). The PAI-1 deficiency did not

affect these bone resorption markers in either placebo- or corticosterone-treated mice

(Figure 6E).

The role of PAI-1 in the apoptosis and proliferation of osteoblasts

Corticosterone treatment increased the number of TUNEL-positive cells on the bone

surface of tibia of WT mice, compared to placebo-treated mice (Figure 7A). The PAI-1

deficiency significantly blunted the corticosterone-induced increase in the number of

TUNEL-positive cells on the bone surface of the tibia of the mice (Figure 7A).

Moreover, an in vitro experiment showed that active PAI-1 significantly increased the


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number of trypan blue-stained and TUNEL-positive MC3T3E1 cells and primary

osteoblasts obtained from mouse calvaria (Figures 7B–E). In contrast, active PAI-1 did

not affect BrdU incorporation into MC3T3E1 cells and into primary osteoblasts

obtained from mouse calvaria (Figures 7F, G), indicating that exogenous PAI-1 did not

affect the proliferation of osteoblasts in vitro. Moreover, the PAI-1 deficiency did not

affect the proliferation impaired by dexamethasone in primary osteoblasts obtained from

mouse calvaria (Figure 7H).

Effects of the GC-induced PAI-1 deficiency on muscle wasting

The qCT analysis revealed that corticosterone treatment significantly reduced muscle

mass in the whole body of WT mice compared to placebo-treated mice (Figures 8A, B).

Corticosterone significantly decreased the tissue weight of the gastrocnemius muscle

but not of the soleus muscle in WT mice (Figures 8C, D). This result was suggestive of

the predominant involvement of type II muscle fibers in the GC-induced muscle

wasting. The PAI-1 deficiency attenuated the corticosterone-induced decrease in tissue

weight of the gastrocnemius muscle in both sexes of mice (Figures 8C, D). Moreover,

the PAI-1 deficiency attenuated the corticosterone-induced decrease in the

cross-sectional area of myofibers in the gastrocnemius muscles in female mice (Figure


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8E and Supplementary Figure 1C). Corticosterone significantly decreased the

expression levels of muscle differentiation-related genes, such as MyoD in the

gastrocnemius muscle tissues of WT female mice. In contrast, corticosterone did not

affect the mRNA levels of myogenin and myosin heavy chain (MHC) in the

gastrocnemius muscle tissue of female mice (Figure 8F). The PAI-1 deficiency

significantly attenuated the corticosterone-induced decrease in the levels of MyoD

mRNA in mice (Figure 8F), suggesting that PAI-1 was involved in muscle

differentiation suppressed by GCs at an early differentiation stage.

DISCUSSION

GC treatment simultaneously induces multiple metabolic disorders in an individual (4).

Therefore, it is important to identify a common therapeutic target for GC-induced

adverse effects. We found that PAI-1 levels are associated with major GC-induced

adverse metabolic effects in mice, for example, insulin resistance, osteoporosis, and

muscle wasting.

Studies suggest that PAI-1 is linked to insulin resistance as well as metabolic

abnormalties (15; 31). We recently reported that PAI-1 deficiency ameliorates insulin

resistance and hyperlipidemia in obese female mice (25). For the first time, we


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demonstrate that PAI-1 deficiency reduces insulin resistance and glucose intolerance but

not hyperlipidemia in GC-treated mice.

GCs impair glucose metabolism by affecting insulin-sensitive organs (the liver,

muscle, and adipose tissue) (3). Several studies revealed that GCs inhibit insulin

signaling in 3T3-L1 adipocytes, L6 myotubes, and HepG2 hepatocytes (30; 32; 33). In

this study, dexamethasone inhibited the phosphorylation of Akt and the glucose uptake,

induced by insulin in 3T3-L1 adipocytes, HepG2 hepatocytes, and C2C12 myotubes in

vitro. Nevertheless, incubation with exogenous PAI-1 suppressed insulin signaling in

HepG2 hepatocytes, but not in 3T3-L1 adipocytes and C2C12 myotubes. Moreover,

active PAI-1 seemed to blunt the levels of key enzymes of gluconeogenesis suppressed

by insulin in HepG2 cells. Thus, PAI-1 participates in GC-induced insulin resistance by

influencing hepatocytes.

Seki et al. reported that dexamethasone increases PAI-1 production in 3T3-L1

adipocytes (34). Our study shows that corticosterone treatment elevates the levels of

PAI-1 mRNA in adipose and muscle tissues of mice. The extent of PAI-1 upregulation is

much higher in adipose tissues than in the muscle tissues. This suggests that circulating

GC-induced PAI-1 produced in adipose and muscle tissues might cause GC-induced

whole-body insulin resistance by impairing insulin signaling in the liver via the


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bloodstream but not in adipose and muscle tissues. The target organs for inhibition of

insulin signaling by GC and PAI-1 seem to be different. PAI-1 circulates in plasma as a

complex with vitronectin, an extracellular matrix glycoprotein (35), stabilizing active

conformation of PAI-1. Lopez-Alemany et al. showed in vitro that PAI-1 inhibits insulin

signaling by competing with integrin αvβ3 for vitronectin binding (36). Possibly PAI-1

deficiency ameliorated GC-induced insulin resistance via enhancement of the

interaction with vitronectin and αvβ3 integrin in the liver. PAI-1 suppression of insulin

signaling in hepatocytes, but not in myotubes and adipocytes, is still unknown. Further

studies are needed to clarify the molecular mechanism underlying PAI-1 effects on

hepatocytes and different PAI-1 tissue sensitivities.

Recent studies suggest that osteoblast-derived osteocalcin, a bone matrix protein, is a

potent regulator of glucose metabolism; acts by modulating the insulin release and

peripheral insulin sensitivity (37; 38). Brennan-Speranza et al. showed that

bone-derived osteocalcin release, which is impaired by GC, is involved in GC-induced

abnormalities in glucose/lipid metabolism (23). Our study demonstrated that the PAI-1

deficiency does not affect levels of carboxylated and uncarboxylated plasma osteocalcin

when suppressed by GC in mice; this deficiency significantly reduces GC-induced

insulin resistance. This suggests that the involvement of PAI-1 in GC-induced insulin


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resistance is independent of osteocalcin in mice.

This study is the first to report that PAI-1 deficiency ameliorates GC-induced bone

loss and decrease in osteoblast numbers in mice. Long-term GC treatment causes bone

loss mainly due to the impaired osteoblastic bone formation (4). GCs decrease the

number of osteoblasts because of the impaired osteoblast differentiation and

proliferation as well as enhanced apoptosis of osteoblasts, resulting in impairment of

bone formation (4). According to our data, corticosterone markedly suppressed levels of

osteogenic genes in the tibia of mice, indicating that GCs impair osteoblast

differentiation. The PAI-1 deficiency did not affect the osteoblast differentiation

suppressed by GC. This suggests that PAI-1 is not linked to the impaired GC-induced

osteoblast differentiation. Also, either endogenous or exogenous PAI-1 does not affect

the proliferation of osteoblasts in vitro. In contrast, the PAI-1 deficiency suppressed

corticosterone-induced apoptosis in the tibia of mice. Moreover, active PAI-1 induced

apoptosis in primary osteoblasts and MC3T3E1 cells. Overall, this indicates that GCs

induce osteopenia through PAI-1 presumably via the enhancement of apoptosis in

osteoblasts.

The enhancement of osteoclastic bone resorption that is induced by excess GC is also

associated with GC-induced bone loss (4). During the initial stage of high-dose GC


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therapy, a rapid but transient increase in bone resorption caused by increase in osteoclast

number and activity can be observed in humans and in animal models (39; 40).

Nonetheless, corticosterone decreased the number of osteoclasts, the levels of RANKL

mRNA, and the RANKL/OPG ratio in the tibia of mice. It is suggested that excess GC

for prolonged period can downregulate osteoclast numbers and function (41).

Furthermore, GC suppresses the formation of osteoclast precursors (41; 42). Therefore,

we can speculate that the suppression of osteoclast formation by GC in this study might

partly be due to the dose and/or duration of GC administration. The PAI-1 deficiency

did not affect either osteoclast numbers or the levels of tibia RANKL and OPG mRNA

(suppressed by the GC), suggesting that PAI-1 is not involved in the changes of

GC-induced bone resorption.

Clinical evidence suggests that GCs negatively affect both muscle and the bone; this

phenomenon may lead to muscle wasting as well as osteoporosis (43-45). Our data

suggest that PAI-1 deficiency blunts decrease in muscle mass and the GC-induced

changes in muscle phenotypes in mice, indicating that PAI-1 is also involved in

GC-induced muscle wasting in mice. Because GCs significantly enhanced endogenous

PAI-1 levels in muscle tissues, GCs might induce muscle wasting in mice through both

muscle’s endogenous PAI-1 and circulating PAI-1.


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GCs directly enhance transactivation of PAI-1 through binding of GR to a

glucocorticoid response element (GRE) in PAI-1’s promoter (46). Several studies

showed that GCs enhance PAI-1 expression in various cell types (35; 47). However, the

source tissue of the PAI-1 production enhanced by GC in vivo has not been identified.

In the present study, GC significantly decreased the levels of PAI-1 mRNA in the liver

and spleen, although the GC elevated PAI-1 mRNA levels in adipose and muscle tissues

in mice. Oishi et al. reported that serum GC concentrations are correlated with PAI-1

expression in adipose tissues but not in the liver of mice (48). In the same study, it was

shown that GC-induced adipose tissue-derived PAI-1 possibly had negative effects on

the GC-induced liver-derived PAI-1 production. Additionally, our previous study

showed that the diabetic state increases PAI-1 expression in the liver of female mice

(16). Thus, we can speculate that there are tissue-specific differences in the PAI-1

induction by GC. The response of different tissue-specific factors related to GC may

negatively affect the PAI-1 induction by GC, depending on an organ. Alternatively, the

change in the metabolic state may modulate the PAI-1 expression in response to GC in

different ways, depending on a tissue.

The reversion of blood glucose levels by the PAI-1 deficiency was not significant at

90 and 120 minutes after insulin injection in male mice (Figure 2F). The blood glucose


24

levels after insulin injection seemed to be higher (not significant) in PAI-1-deficient

mice than in the wild-type mice, suggesting that slight sex difference might exist in

PAI-1 involvement in the pathogenesis of GC-induced insulin resistance in mice. We

previously reported that PAI-1 is involved in the pathogenesis of type 1 diabetic

osteoporosis in female mice but not in male mice (16). Studies have suggested that a

protein linked to the sex chromosomes is associated with the sex differences in the

prevalence of metabolic abnormality and osteoporosis (49; 50). This suggests that a

protein linked to a sex chromosome might be responsible for the sex differences

observed in the present study. Further studies are needed to clarify the above issues.

PAI-1 is considered as a humoral factor that affects systemic metabolism. Our

previous study suggested that a type 1 diabetic state increases PAI-1 expression in the

liver, resulting in increased circulating PAI-1 levels in female mice (16). Upregulation

of circulating PAI-1 impairs osteoblast differentiation and mineralization, and promotes

adipogenesis in bone tissues; the latter effect leads to diabetic osteoporosis. Moreover,

PAI-1 is involved in obesity-induced insulin resistance and hyperlipidemia but not bone

loss in female mice, although the role of PAI-1 in bone metabolism in the type 2

diabetic state remains completely unknown at present (25). However, GC increases

PAI-1 production mainly in adipose tissues, thereby leading to increased blood PAI-1


25

levels. The upregulation of circulating PAI-1 may lead to osteoporosis as well as insulin

resistance and muscle wasting induced by GC (Figure 8G). Thus, PAI-1 possibly plays

an important role as a humoral factor to regulate various metabolic states in presence of

excess GC as well as in diabetes.

In conclusion, for the first time, we demonstrated that PAI-1 is involved in the

metabolic adverse effects of GC treatment, for example, in insulin resistance, bone loss,

and muscle wasting in mice. PAI-1 may be a novel therapeutic target that can help to

decrease the risk of GC-induced adverse outcomes. PAI-1 may serve as a diagnostic

marker of GC-induced diabetes, osteoporosis, and muscle wasting.

ACKNOWLEDGMENTS

This study was supported by grants-in-aid 26860152 and 24590289 from the Ministry

of Science, Education, and Culture of Japan (to Y.T. and H.K., respectively), a grant

from Japan Osteoporosis Foundation, a grant from Takeda Science Foundation, and a

grant from Kinki University.

There are no potential conflicts of interest relevant to this article.


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Y.T. researched data, contributed to the discussion, wrote the manuscript, reviewed, and

edited the manuscript. N.K., K.Oka., M.Y., K.Oku., Y.C. and O.M. contributed to the

discussion, reviewed, and edited the manuscript. H.K. contributed to the discussion,

wrote the manuscript, reviewed, and edited the manuscript. H.K. 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.

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31

FIGURE LEGENDS

FIG. 1. Effects of glucocorticoid (GC) on the levels of circulating PAI-1 and on

PAI-1 expression in mice

The growth curve during experiments (A, B), the percentage of fat mass (C, D), and

visceral and subcutaneous fat mass (E, F) in placebo-treated (Cont) WT,

corticosterone-treated (CS) WT, Cont PAI-1 knockout (KO), and CS PAI-1 KO female

and male mice. ##

P < 0.01 versus Cont WT mice, **P < 0.01 versus Cont KO mice (n =

11 and 6 in each group of female and male mice, respectively). The levels of plasma

PAI-1 (G, H) in both sexes of Cont and CS WT mice. **P < 0.01 (n = 11 and 6 in each

group of female and male mice, respectively). The levels of PAI-1 mRNA in white

adipose tissues (WAT), liver, muscle, tibia, heart, lung, kidney, and spleen (I) in Cont

and CS WT female mice. **P < 0.01 (n = 5 in each group). The results are shown

relative to β-actin mRNA values (mean ± SEM). Two-way repeated-measures ANOVA

and 2-way ANOVA (2WA) were used for growth curve (A, B) and for fat mass (C-F),

respectively, to compare the drug effects of (d) placebo and the corticosterone pellets

(Cont and CS) on both the genotypes of mice (g) (PAI-1 WT and PAI-1 KO). When

significant differences were observed among individual means, post hoc analysis was

carried out using Tukey’s test. For assessment of body fat composition using a


32

quantitative computed tomography (qCT) scan, contiguous 2-mm slice images between

L1 and L5 were used for quantitative assessment in the LaTheta software (version 3.40).

Visceral fat and subcutaneous fat were identified, and the total fat content, visceral-fat

weight, and subcutaneous-fat weight were calculated from all slice images and were

evaluated quantitatively. The percentage of fat mass was calculated by dividing total fat

mass by total body mass.

FIG. 2. Effects of the PAI-1 deficiency on glucocorticoid (GC)-induced abnormal

glucose metabolism in female mice

The fasting levels of blood glucose (A, B) and plasma insulin (C, D) in placebo-treated

(Cont) WT, corticosterone-treated (CS) WT, Cont PAI-1 knockout (KO), and CS PAI-1

KO female and male mice. The results are presented as mean ± SEM. **P < 0.01 (n =

11 and 6 in each group of female and male mice, respectively). Responses of blood

glucose to a single intraperitoneal (i.p.) injection of insulin (E, F) in Cont WT (open

circles), CS WT (open squares), Cont PAI-1 KO (closed circles), and CS PAI-1 KO

(closed squares) female mice (n = 5 and 6 in each group of female and male mice,

respectively). Responses of blood glucose levels (G, H) to a single i.p. injection of

glucose in Cont WT (open circles), CS WT (open squares), Cont PAI-1 KO (closed


33

circles), and CS PAI-1 KO (closed squares) female and male mice (n = 11 and 6 in each

group of female and male mice, respectively). Responses of plasma insulin (I, J) to a

single i.p. injection of glucose in Cont WT (open circles), CS WT (open squares), Cont

PAI-1 KO (closed circles), and CS PAI-1 KO (closed squares) female and male mice; (n

= 6 and 6 in each group of female and male mice, respectively). The results are

presented as mean ± SEM. *P < 0.05, **P < 0.01 versus CS WT mice. Two-way

ANOVA (2WA) and 2-way repeated-measures ANOVA were used for A-D and for E-J,

respectively, to compare the drug effects (d) of a placebo and a corticosterone pellet

(Cont and CS) on the mice of 2 genotypes (g) (PAI-1 WT and PAI-1 KO). When

significant differences were observed, individual means of the bars were compared

using Tukey’s post hoc test.

FIG. 3. The influence of PAI-1 and dexamethasone on insulin signaling in

hepatocytes, myocytes, and adipocytes in vitro

Phosphorylation of Akt, stimulated by insulin treatment (100 nM) for 15 min with or

without active PAI-1 (20 nM) and dexamethasone (Dex, 100 nM) treatment for 24 h in

HepG2 cells (A), differentiated C2C12 cells (B), and differentiated 3T3-L1 cells (C).

2-Deoxyglucose uptake [the concentration of 2-deoxyglucose-6-phosphate (2DG6P)]


34

stimulated by insulin treatment (1 µM) for 20 min with or without active PAI-1 (20 nM)

and Dex (1 µM) treatment for 24 h in HepG2 cells (D), differentiated C2C12 cells (E),

and differentiated 3T3-L1 cells (F). The results are presented as mean ± SEM of 3

experiments; *P < 0.05, **P < 0.01. Tukey’s post hoc test was used. The mRNA levels

of G6Pase (G) and PEPCK (H) in HepG2 cells incubated with or without active PAI-1

(20 nM) and dexamethasone (Dex, 100 nM) in the presence of as well as without insulin

(1 nM) for 24 h. The results are presented as mean ± SEM of 6 experiments; *P < 0.05,

**P < 0.01. Tukey’s post hoc test was used

FIG. 4. Effects of the PAI-1 deficiency on hyperlipidemia induced by

glucocorticoid (GC) in mice

The levels of plasma triglycerides (TG) (A, B) and total cholesterol (T-Chol) (C, D) in

placebo-treated (Cont) WT, corticosterone-treated (CS) WT, Cont PAI-1 knockout (KO),

and CS PAI-1 KO female and male mice. The results are shown as mean ± SEM (n = 11

and 6 in each group of female and male mice, respectively). Two-way ANOVA (2WA)

was used to compare the drug effects (d) of the placebo and corticosterone pellet (Cont

and CS) on the mice of 2 genotypes (g) (PAI-1 WT and PAI-1 KO). When significant

differences were observed, individual means of the bars were compared using Tukey’s


35

post hoc test.

FIG. 5. Effects of the PAI-1 deficiency on bone loss induced by glucocorticoid (GC)

treatment in mice

BMD values in total (A, B), trabecular (C, D), and cortical bones (E, F), bone volume

fraction (BV/TV) (G, H), cortical thickness (I, J), cortical area (K, L), total

cross-sectional area (M, N), and trabecular area (O, P) of the tibia in placebo-treated

(Cont) WT, corticosterone-treated (CS) WT, Cont PAI-1 knockout (KO), and CS PAI-1

KO female and male mice. The results are presented as mean ± SEM; *P < 0.05, **P <

0.01 (n = 11 and 6 in each group of female and male mice, respectively). Two-way

ANOVA (2WA) was used to compare the drug effects (d) of the placebo and

corticosterone pellet (Cont and CS) on the mice of 2 genotypes (g) (PAI-1 WT and

PAI-1 KO). When significant differences were observed, individual means of the bars

were compared using Tukey’s post hoc test. For assessment of trabecular BMD, total

bone volume, trabecular bone volume, and bone trabecular area, trabecular regions of

interest (ROIs) extended from 96 um distal to the end of the proximal growth plate over

1.5 mm toward the diaphysis. For assessment of cortical BMD, thickness and area,

ROIs were defined as 2.0-mm segments of the mid-diaphysis tibia. For assessment of


36

total BMD and total cross-sectional area, ROIs were defined as 9,600-µm segments

(100 slices) from the distal end of the proximal growth plate of the tibia. The bone

volume fraction was calculated as the ratio of trabecular bone volume (BV [mm3]) to

the total bone volume (TV [mm3]). Parameters used for the CT scans were as follows:

tube voltage, 50 kVp; tube current, 500 µA; integration time, 3.6 ms; axial field of view,

48 mm; and voxel size of 48 × 96 µm with a slice thickness of 96 µm. Bone parameters

were analyzed using the LaTheta software (version 3.40).

FIG. 6. Effects of the PAI-1 deficiency on abnormal bone metabolism induced by

GC treatment in female mice

The levels of expression of Runx2, Osterix, Alp, Ocn, and Col1 mRNA in the tibia (A),

plasma osteocalcin (OCN) (B), and plasma uncarboxylated OCN (ucOCN) (C) in

placebo-treated (Cont) WT (n = 5), corticosterone-treated (CS) WT (n = 5), Cont PAI-1

knockout (KO; n = 5), and CS PAI-1 KO (n = 5) female mice. The number of

ALP-positive cells on the bone surface of tibia in Cont WT (n = 5), CS WT (n = 5),

Cont PAI-1 KO (n = 5), and CS PAI-1 KO (n = 5) female mice (D). The levels of

RANKL and OPG mRNA in tibia, and RANKL/OPG ratio, plasma cross-linked

C-telopeptide of type I collagen (CTX), and the number of tartrate-resistant acid


37

phosphatase (TRAP)-positive multinucleated cells in the tibia of Cont WT (n = 5), CS

WT (n = 5), Cont PAI-1 KO (n = 5), and CS PAI-1 KO (n = 5) female mice (E). The

results are shown as mean ± SEM; **P < 0.01. The results of mRNA expression are

shown relative to β-actin mRNA values. Two-way ANOVA (2WA) was used to compare

the drug effects (d) of the placebo and corticosterone pellet (Cont and CS) on the mice

of 2 genotypes (g) (PAI-1 WT and PAI-1 KO). When significant differences were

observed, individual means of the bars were compared by means of Tukey’s post hoc

test.

FIG. 7. The influence of PAI-1 on apoptosis and proliferation of osteoblasts

The number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling

(TUNEL)-positive cells on the bone surface in the tibia of placebo-treated (Cont) WT (n

= 5), corticosterone-treated (CS) WT (n = 5), Cont PAI-1 knockout (KO; n = 5), and CS

PAI-1 KO (n = 5) female mice (A). Two-way ANOVA (2WA) was used to compare the

drug effects (d) of the placebo and corticosterone pellet (Cont and CS) on the mice of 2

genotypes (g) (PAI-1 WT and PAI-1 KO). When significant differences were observed,

individual means of the bars were compared using Tukey’s post hoc test. The cell death

rate in primary osteoblasts (OB) from female mouse calvaria (B) and MC3T3E1 cells


38

(C) incubated with or without active PAI-1 (20 nM) for 24 h. Cell death was identified

by trypan blue staining. The number of apoptotic primary OBs from female mouse

calvaria (D) and MC3T3E1 (E) cells incubated with or without active PAI-1 (20 nM) for

24 h. Apoptotic cells were identified by TUNEL staining. The results are shown as mean

± SEM of 3 experiments; **P < 0.01. Unpaired t-test was used. Proliferation of primary

OBs from WT female-mouse calvaria (F) and MC3T3E1 cells (G) incubated with or

without active PAI-1 (20 nM) for 24 h. Proliferation of primary OBs from female WT

and PAI-1 KO mouse calvaria incubated with or without dexamethasone (Dex; 100 nM)

(H). Cell proliferation was assessed using a bromodeoxyuridine (BrdU) incorporation

assay. The results are shown as mean ± SEM of 3 experiments. Two-way ANOVA

(2WA) was used to compare the drug effects (d) of vehicle and Dex (Cont and Dex) on

the mice of 2 genotypes (g) (PAI-1 WT and PAI-1 KO). When significant differences

were observed, individual means of the bars were compared using Tukey’s post hoc test.

FIG. 8. Effects of the PAI-1 deficiency on muscle wasting induced by glucocorticoid

(GC) treatment in mice

Total muscle mass (A, B) in placebo-treated (Cont) WT, corticosterone-treated (CS) WT,

Cont PAI-1 knockout (KO), and CS PAI-1 KO female and male mice. The results are


39

shown as mean ± SEM; *P < 0.05, **P < 0.01 (n = 11 and 6 in each group of female and

male mice, respectively). Tissue weight of the gastrocnemius muscle (GAS) and soleus

muscle (C, D) in Cont WT, CS WT, Cont PAI-1 KO, and CS PAI-1 KO female and male

mice. Cross-sectional area (CSA) of a myofiber (E), and the mRNA levels of MyoD,

myogenin, and myosin heavy chain (MHC) (F) in the gastrocnemius muscle in Cont WT,

CS WT, Cont PAI-1 KO, and CS PAI-1 KO female mice. For assessment of the

cross-sectional area of a myofiber, a minimum of 1000 myofibers per muscle were

analyzed on a hematoxylin and eosin-stained gastrocnemius muscle slice. The results

are presented as mean ± SEM; *P < 0.05, **P < 0.01 (n = 6 in each group of female and

male mice, respectively). Two-way ANOVA (2WA) was used to compare the drug

effects (d) of the placebo and corticosterone pellet (Cont and CS) on the mice of 2


individual means of the bars were compared using Tukey’s post hoc test. For assessment

of total muscle mass using a qCT scan, contiguous 2-mm slice images between L1 and

the toe were used for quantitative assessment in the LaTheta software (version 3.40).

The proposed hypothesis for the role of PAI-1 in the pathogenesis of insulin resistance,

bone loss, and muscle wasting induced by GC in mice (G): GC increases PAI-1

expression in adipose tissue, resulting in elevation of circulating PAI-1 levels in mice.


40

An elevated level of circulating PAI-1 induces insulin resistance in the liver, bone loss

due to the increased apoptosis of osteoblasts, and muscle wasting in mice.


FIG. 1. Effects of a glucocorticoid (GC) on the levels of circulating PAI-1 and on PAI-1 expression in mice The growth curve during experiments (A, B), the percentage of fat mass (C, D), and visceral and

subcutaneous fat mass (E, F) in placebo-treated (Cont) WT, corticosterone-treated (CS) WT, Cont PAI-1

knockout (KO), and CS PAI-1 KO female and male mice. ##P < 0.01 versus Cont WT mice, **P < 0.01 versus Cont KO mice (n = 11 and 6 in each group of female and male mice, respectively). The levels of plasma PAI-1 (G, H) in both sexes of Cont and CS WT mice. **P < 0.01 (n = 11 and 6 in each group of female and male mice, respectively). The levels of PAI-1 mRNA in white adipose tissues (WAT), liver,

muscle, tibia, heart, lung, kidney, and spleen (I) in Cont and CS WT female mice. **P < 0.01 (n = 5 in each group). The results are shown relative to β-actin mRNA values (mean ± SEM). Two-way repeated-measures ANOVA and 2-way ANOVA (2WA) were used for growth curve (A, B) and for fat mass (C-F), respectively, to compare the drug effects of (d) placebo and the corticosterone pellets (Cont and CS) on both the genotypes of mice (g) (PAI-1 WT and PAI-1 KO); when significant differences were observed among individual means,

post hoc analysis was carried out using Tukey’s test. For assessment of body fat composition using a quantitative computed tomography (qCT) scan, contiguous 2-mm slice images between L1 and L5 were used

for quantitative assessment in the LaTheta software (version 3.40). Visceral fat and subcutaneous fat were identified, and the total fat content, visceral-fat weight, and subcutaneous-fat weight were calculated from all slice images and were evaluated quantitatively. The percentage of fat mass was calculated by dividing

total fat mass by total body mass. 173x171mm (300 x 300 DPI)



FIG. 2. Effects of the PAI-1 deficiency on glucocorticoid (GC)-induced abnormal glucose metabolism in female mice

The fasting levels of blood glucose (A, B) and plasma insulin (C, D) in placebo-treated (Cont) WT,

corticosterone-treated (CS) WT, Cont PAI-1 knockout (KO), and CS PAI-1 KO female and male mice. The results are presented as mean ± SEM. **P < 0.01 (n = 11 and 6 in each group of female and male mice, respectively). Responses of blood glucose to a single intraperitoneal (i.p.) injection of insulin (E, F) in Cont WT (open circles), CS WT (open squares), Cont PAI-1 KO (closed circles), and CS PAI-1 KO (closed squares) female mice (n = 5 and 6 in each group of female and male mice, respectively). Responses of blood glucose levels (G, H) to a single i.p. injection of glucose in Cont WT (open circles), CS WT (open squares), Cont PAI-1 KO (closed circles), and CS PAI-1 KO (closed squares) female and male mice (n = 11 and 6 in each group of female and male mice, respectively). Responses of plasma insulin (I, J) to a single i.p. injection of glucose in Cont WT (open circles), CS WT (open squares), Cont PAI-1 KO (closed circles), and CS PAI-1 KO (closed squares) female and male mice; (n = 6 and 6 in each group of female and male mice, respectively). The results are presented as mean ± SEM. *P < 0.05, **P < 0.01 versus CS WT mice. Two-way ANOVA (2WA)

and 2-way repeated-measures ANOVA were used for A-D and for E-J, respectively, to compare the drug effects (d) of a placebo and a corticosterone pellet (Cont and CS) on the mice of 2 genotypes (g) (PAI-1 WT and PAI-1 KO). When significant differences were observed, individual means of the bars were compared

using Tukey’s post hoc test.


188x203mm (300 x 300 DPI)


FIG. 3. The influence of PAI-1 and dexamethasone on insulin signaling in hepatocytes, myocytes, and adipocytes in vitro

Phosphorylation of Akt, stimulated by insulin treatment (100 nM) for 15 min with or without active PAI-1 (20 nM) and dexamethasone (Dex, 100 nM) treatment for 24 h in HepG2 cells (A), differentiated C2C12 cells (B), and differentiated 3T3-L1 cells (C). 2-Deoxyglucose uptake (the concentration of 2-deoxyglucose-6-

phosphate (2DG6P)) stimulated by insulin treatment (1 µM) for 20 min with or without active PAI-1 (20 nM) and Dex (1 µM) treatment for 24 h in HepG2 cells (D), differentiated C2C12 cells (E), and differentiated 3T3-

L1 cells (F). The results are presented as mean ± SEM of 3 experiments; *P < 0.05, **P < 0.01. Tukey’s

post hoc test was used. The mRNA levels of G6Pase (G) and PEPCK (H) in HepG2 cells incubated with or without active PAI-1 (20 nM) and dexamethasone (Dex, 100 nM) in the presence of as well as without

insulin (1 nM) for 24 h. The results are presented as mean ± SEM of 6 experiments; *P < 0.05, **P < 0.01. Tukey’s post hoc test was used. 179x184mm (300 x 300 DPI)


FIG. 4. Effects of the PAI-1 deficiency on hyperlipidemia induced by glucocorticoid (GC) in mice The levels of plasma triglycerides (TG) (A, B) and total cholesterol (T-chol) (C, D) in placebo-treated (Cont) WT, corticosterone-treated (CS) WT, Cont PAI-1 knockout (KO), and CS PAI-1 KO female and male mice. The results are shown as mean ± SEM; **P < 0.01 (n = 11 and 6 in each group of female and male mice,

respectively). Two-way ANOVA (2WA) was used to compare the drug effects (d) of the placebo and corticosterone pellet (Cont and CS) on the mice of 2 genotypes (g) (PAI-1 WT and PAI-1 KO). When

significant differences were observed, individual means of the bars were compared using Tukey’s post hoc test.

83x39mm (300 x 300 DPI)


FIG. 5. Effects of the PAI-1 deficiency on bone loss induced by glucocorticoid (GC) treatment in mice BMD values in total (A, B), trabecular (C, D), and cortical bones (E, F), bone volume fraction (BV/TV) (G, H), cortical thickness (I, J), cortical area (K, L), total cross-sectional area (M, N), and trabecular area (O, P) of the tibia in placebo-treated (Cont) WT, corticosterone-treated (CS) WT, Cont PAI-1 knockout (KO), and CS PAI-1 KO female and male mice. The results are presented as mean ± SEM; *P < 0.05, **P < 0.01 (n = 11 and 6 in each group of female and male mice, respectively). Two-way ANOVA (2WA) was used to compare the drug effects (d) of the placebo and corticosterone pellet (Cont and CS) on the mice of 2 genotypes (g) (PAI-1 WT and PAI-1 KO). When significant differences were observed, individual means of the bars were

compared using Tukey’s post hoc test. For assessment of trabecular BMD, total bone volume, trabecular bone volume, and bone trabecular area, trabecular regions of interest (ROIs) extended from 96 um distal to the end of the proximal growth plate over 1.5 mm toward the diaphysis. For assessment of cortical BMD, thickness and area, ROIs were defined as 2.0-mm segments of the mid-diaphysis tibia. For assessment of total BMD and total cross-sectional area, ROIs were defined as 9,600-µm segments (100 slices) from the


distal end of the proximal growth plate of the tibia. The bone volume fraction was calculated as the ratio of trabecular bone volume (BV [mm3]) to the total bone volume (TV [mm3]). Parameters used for the CT scans were as follows: tube voltage, 50 kVp; tube current, 500 µA; integration time, 3.6 ms; axial field of view, 48 mm; and voxel size of 48 × 96 µm with a slice thickness of 96 µm. Bone parameters were analyzed

using the LaTheta software (version 3.40).

208x249mm (300 x 300 DPI)


FIG. 6. Effects of the PAI-1 deficiency on abnormal bone metabolism induced by GC treatment in female mice

The levels of expression of Runx2, Osterix, Alp, Ocn, and Col1 mRNA in the tibia (A), plasma osteocalcin

(OCN) (B), and plasma uncarboxylated OCN (ucOCN) (C) in placebo-treated (Cont) WT (n = 5), corticosterone-treated (CS) WT (n = 5), Cont PAI-1 knockout (KO; n = 5), and CS PAI-1 KO (n = 5) female mice. The number of ALP-positive cells on the bone surface of tibia in Cont WT (n = 5), CS WT (n = 5), Cont PAI-1 KO (n = 5), and CS PAI-1 KO (n = 5) female mice (D). The levels of RANKL and OPG mRNA in tibia,

and RANKL/OPG ratio, plasma cross-linked C-telopeptide of type I collagen (CTX), and the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in the tibia of Cont WT (n = 5), CS WT (n = 5), Cont PAI-1 KO (n = 5), and CS PAI-1 KO (n = 5) female mice (E). The results are shown as

mean ± SEM; **P < 0.01. The results of mRNA expression are shown relative to β-actin mRNA values. Two-way ANOVA (2WA) was used to compare the drug effects (d) of the placebo and corticosterone pellet (Cont

and CS) on the mice of 2 genotypes (g) (PAI-1 WT and PAI-1 KO). When significant differences were observed, individual means of the bars were compared by means of Tukey’s post hoc test.

170x166mm (300 x 300 DPI)


FIG. 7. The influence of PAI-1 on apoptosis and proliferation of osteoblasts The number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells on the bone surface in the tibia of placebo-treated (Cont) WT (n = 5), corticosterone-treated (CS) WT (n = 5), Cont PAI-1 knockout (KO; n = 5), and CS PAI-1 KO (n = 5) female mice (A). Two-way ANOVA (2WA)

was used to compare the drug effects (d) of the placebo and corticosterone pellet (Cont and CS) on the mice of 2 genotypes (g) (PAI-1 WT and PAI-1 KO). When significant differences were observed, individual means of the bars were compared using Tukey’s post hoc test. The cell death rate in primary osteoblasts (OB) from female mouse calvaria (B) and MC3T3E1 cells (C) incubated with or without active PAI-1 (20 nM) for 24 h.

Cell death was identified by trypan blue staining. The number of apoptotic primary OBs from female mouse calvaria (D) and MC3T3E1 (E) cells incubated with or without active PAI-1 (20 nM) for 24 h. Apoptotic cells were identified by TUNEL staining. The results are shown as mean ± SEM of 3 experiments; **P < 0.01. Unpaired t-test was used. Proliferation of primary OBs from WT female-mouse calvaria (F) and MC3T3E1 cells (G) incubated with or without active PAI-1 (20 nM) for 24 h. Proliferation of primary OBs from female

WT and PAI-1 KO mouse calvaria incubated with or without dexamethasone (Dex; 100 nM) (H). Cell proliferation was assessed using a bromodeoxyuridine (BrdU) incorporation assay. The results are shown as mean ± SEM of 3 experiments. Two-way ANOVA (2WA) was used to compare the drug effects (d) of vehicle

and Dex (Cont and Dex) on the mice of 2 genotypes (g) (PAI-1 WT and PAI-1 KO). When significant differences were observed, individual means of the bars were compared using Tukey’s post hoc test.

133x101mm (300 x 300 DPI)


FIG. 8. Effects of the PAI-1 deficiency on muscle wasting induced by glucocorticoid (GC) treatment in mice Total muscle mass (A, B) in placebo-treated (Cont) WT, corticosterone-treated (CS) WT, Cont PAI-1

knockout (KO), and CS PAI-1 KO female and male mice. The results are shown as mean ± SEM; *P < 0.05,

**P < 0.01 (n = 11 and 6 in each group of female and male mice, respectively). Tissue weight of the gastrocnemius muscle (GAS) and soleus muscle (C, D) in Cont WT, CS WT, Cont PAI-1 KO, and CS PAI-1 KO

female and male mice. Cross-sectional area (CSA) of a myofiber (E), and the mRNA levels of MyoD, myogenin, and myosin heavy chain (MHC) (F) in the gastrocnemius muscle in Cont WT, CS WT, Cont PAI-1 KO, and CS PAI-1 KO female mice. For assessment of the cross-sectional area of a myofiber, a minimum of 1000 myofibers per muscle were analyzed on a hematoxylin and eosin-stained gastrocnemius muscle slice. The results are presented as mean ± SEM; *P < 0.05, **P < 0.01 (n = 6 in each group of female and male mice, respectively). Two-way ANOVA (2WA) was used to compare the drug effects (d) of the placebo and corticosterone pellet (Cont and CS) on the mice of 2 genotypes (g) (PAI-1 WT and PAI-1 KO). When

significant differences were observed, individual means of the bars were compared using Tukey’s post hoc test. For assessment of total muscle mass using a qCT scan, contiguous 2-mm slice images between L1 and

the toe were used for quantitative assessment in the LaTheta software (version 3.40). The proposed hypothesis for the role of PAI-1 in the pathogenesis of insulin resistance, bone loss, and muscle wasting induced by GC in mice (G): GC increases PAI-1 expression in adipose tissue, resulting in elevation of

circulating PAI-1 levels in mice. An elevated level of circulating PAI-1 induces insulin resistance in the liver, bone loss due to the increased apoptosis of osteoblasts, and muscle wasting in mice.

138x109mm (300 x 300 DPI)


SUPPLEMENTARY DATA

Supplementary Figure 1. The influence of glucocorticoid (GC) treatment and the

PAI-1 deficiency on lipid accumulation in adipose, liver, and muscle tissues of

female mice.

A representative picture of the adipose tissue (A), liver (B), and muscle tissue (C) of

placebo-treated (Cont) WT, corticosterone-treated (CS) WT, Cont PAI-1 knockout (KO),

and CS PAI-1 KO female mice (bar: 50 µm). Triglyceride content in the liver (D) and

gastrocnemius muscle tissue (E) of Cont WT, CS WT, Cont PAI-1 KO, and CS PAI-1

KO female mice. The results are presented as mean ± SEM; *P < 0.05, **P < 0.01 (n =

6 in each group of female mice). Two-way ANOVA (2WA) was used to compare the

drug effects (d) of the placebo and corticosterone pellet (Cont and CS) on the mice of 2


individual means of the bars were compared using Tukey’s post hoc test.


PAI-1: plasminogen activator inhibitor 1, RANKL: receptor activator of nuclear

factor-кB ligand, MHC: myosin heavy chain, GAPDH: glyceraldehyde 3-phosphate

dehydrogenase.

Supplementary Table 1. Primer used for real-time PCR

Primer Sequence

Mouse

PAI-1 Forward 5’-TTCAGCCCTTGCTTGCCTC-3’

PAI-1 Reverse 5’-ACACTTTTACTCCGAAGTCGGT-3'

Runx2 Forward 5'-AAATGCCTCCGCTGTTATGAA-3'

Runx2 Reverse 5'-GCTCCGGCCCACAAATCT-3'

Osterix Forward 5’-AGCGACCACTTGAGCAAACAT-3’

Osterix Reverse 5’-GCGGCTGATTGGCTTCTT-3’

Alkaline phosphatase Forward 5’-ATCTTTGGTCTGGCTCCCATG-3’

Alkaline phosphatase Reverse 5’-TTTCCCGTTCACCGTCCAC-3’

Osteocalcin Forward 5’-CCTGAGTCTGACAAAGCCTTCA-3’

Osteocalcin Reverse 5’-GCCGGAGTCTGTTCACTACCTT-3’

Type 1 collagen Forward 5’-AACCCTGCCCGCACATG-3’

Type 1 collagen Reverse 5’-CAGACGGCTGAGTAGGGAACA’-3

RANKL Forward 5'-CACAGCGCTTCTCAGGAGCT-3'

RANKL Reverse 5'-CATCCA ACCATGAGCCTTCC-3'

Osteoprotegerin Forward 5’-CTTGGGTCTGTTGCTTGGTGA-3’

Osteoprotegerin Reverse 5’-GCCGCTTCCTTACACACCAG-3’

MyoD Forward 5’-AGCACTACAGTGGCGACTCAG-3’

MyoD Reverse 5’-AGGCGGTGTCGTAGCCATTC-3’

Myogenin Forward 5’-GCTGCCTAAAGTGGAGATCCT-3’

Myogenin Reverse 5’-GCGCTGTGGGAGTTGCAT-3’

MHC Forward 5’-CTCTTCCCGCTTTGGTAAGTT-3’

MHC Reverse 5’-CAGGAGCATTTCGATTAGATCCG-3’

β-actin Forward 5’-TACCACAGGCATTGTGATGG-3’

β-actin Reverse 5’-TTTGATGTCACGCACGATTT-3’

Human

Glucose-6-phosphatase Forward 5’-TCATCTTGGTGTCCGTGATCG-3’

Glucose-6-phosphatase Reverse 5’-TTTATCAGGGGCACGGAAGTG-3’

PEPCK Forward 5’-GAAAAAACCTGGGGCACAT-3’

PEPCK Reverse 5’-TTGCTTCAAGGCAAGGATCTCT-3’

GAPDH Forward 5’-ACCACAGTCCATGCCATCAC-3’

GAPDH Reverse 5’-TCCACCACCCTGTTGCTGTA-3’


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