Yukinori Tamura,1 Naoyuki Kawao,1 Masato Yano,1 Kiyotaka Okada,1

Katsumi Okumoto,2 Yasutaka Chiba,3 Osamu Matsuo,4 and Hiroshi Kaji1

Role of PlasminogenActivator Inhibitor-1in Glucocorticoid-Induced Diabetesand Osteopenia in MiceDiabetes 2015;64:2194–2206 | DOI: 10.2337/db14-1192

Long-term use of glucocorticoids (GCs) causes numer-ous adverse effects, including glucose/lipid abnormali-ties, osteoporosis, and muscle wasting. The pathogenicmechanism, however, is not completely understood. Inthis study, we used plasminogen activator inhibitor-1(PAI-1)–deficient mice to explore the role of PAI-1 in GC-induced glucose/lipid abnormalities, osteoporosis, andmuscle wasting. Corticosterone markedly increased thelevels of circulating PAI-1 and the PAI-1 mRNA level inthe white adipose tissue of wild-type mice. PAI-1 defi-ciency significantly reduced insulin resistance and glu-cose intolerance but not hyperlipidemia induced by GC.An in vitro experiment revealed that active PAI-1 treat-ment inhibits insulin-induced phosphorylation of Akt andglucose uptake in HepG2 hepatocytes. However, thiswas not observed in 3T3-L1 adipocytes and C2C12 my-otubes, indicating that PAI-1 suppressed insulin signalingin hepatocytes. PAI-1 deficiency attenuated the GC-induced bone loss presumably via inhibition of apoptosisof osteoblasts. Moreover, the PAI-1 deficiency also pro-tected from GC-induced muscle loss. In conclusion, thecurrent 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 ther-apeutic target to mitigate the adverse effects of GC.

Glucocorticoids (GCs) have strong anti-inflammatoryproperties and are highly effective in the treatment ofallergies and inflammatory and autoimmune conditions,such as rheumatoid arthritis, asthma, inflammatory boweldisease, and collagen diseases (1,2). Despite the high

efficacy of GC treatment, its clinical use is limited due toadverse effects, such as diabetes, hyperlipidemia, osteo-porosis, muscle wasting, and immunosuppression, whichdepend on the administered dose and duration of GCtreatment (3–5).

GC-induced diabetes and osteoporosis are manifesta-tions of adverse metabolic effects because of the highincidence. A clinical study showed that .11% of GC usersdevelop diabetes within 3 years of GC therapy (6), anda larger number of patients transition into a prediabeticstate, such as insulin resistance and impaired glucose tol-erance. In muscles, GCs have been shown to suppressa number of steps in the insulin signaling network (3,7).GCs directly promote hepatic gluconeogenesis, leading tohyperglycemia (7). Moreover, GCs promote proteolysis, li-polysis, free fatty acid production, and fat accumulation inthe liver that contributes to insulin resistance.

GC treatment is the most common cause of secondaryosteoporosis (8). GCs affect osteoblasts, osteoclasts, andosteocytes. Osteoblasts are generally considered the mainskeletal target (4,9). Several studies suggest that GCs sup-press bone formation by inhibiting differentiation, pro-liferation, and apoptosis of osteoblasts, leading toosteoporosis (4,9); however, the mechanism is not wellunderstood. In addition, the patients treated with GCfrequently suffer from muscle wasting (10). GCs are alsoimportant mediators of muscle wasting in many patho-logical conditions, such as sepsis, cachexia, starvation, andmetabolic acidosis (11).

Experimental evidence suggests that the above-mentionedeffects of GCs are induced in the cytoplasm by the direct

1Department of Physiology and Regenerative Medicine, Kinki University Faculty ofMedicine, Osakasayama, Japan2Life Science Research Institute, Kinki University, Osakasayama, Japan3Clinical Research Center, Kinki University Hospital, Osakasayama, Japan4Kinki University Faculty of Medicine, Osakasayama, Japan

Corresponding author: Hiroshi Kaji, hkaji@med.kindai.ac.jp.

Received 31 July 2014 and accepted 20 December 2014.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db14-1192/-/DC1.

© 2015 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

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PATHOPHYSIO

LOGY

action of GC binding to GC receptors (4). Nonetheless, theprecise mechanisms are not fully understood, and theevidence of systemic mediators, in the adverse effects ofGCs, is lacking.

Plasminogen activator inhibitor-1 (PAI-1) is a serineprotease inhibitor that primarily inhibits tissue-typeand urokinase-type plasminogen activators; hence, it isan inhibitor of fibrinolysis. PAI-1 is a well-knownadipocytokine, being upregulated along with fat accu-mulation (12). It has been suggested that elevatedlevels of circulating PAI-1 is a risk factor in cardio-vascular diseases (atherosclerosis), obesity, and diabe-tes (13–15). Moreover, we recently demonstrated infemale mice with streptozotocin-induced type 1 diabe-tes that PAI-1 is involved in bone loss (16). PAI-1 isupregulated in atrophic skeletal muscle (17), and thischange is associated with impaired muscle regenera-tion (18,19). Several clinical studies showed that circu-lating PAI-1 concentration is elevated in patients withCushing syndrome or during corticosteroid treatment(20,21). Nonetheless, the role of PAI-1 in GC-inducedglucose/lipid abnormalities, osteoporosis, and musclewasting is unknown. Therefore, we examined the ef-fects of PAI-1 deficiency on GC-induced glucose/lipidand bone metabolism abnormalities as well as musclewasting in mice.

RESEARCH DESIGN AND METHODS

Animal ExperimentsForty-four female and 24 male mice with a mixed C57BL/6J(81.25%) and 129/SvJ (18.75%) genetic backgroundwere analyzed as described in the figure legends. Weincluded 22 female and 12 male mice with PAI-1 genedeficiency (PAI-1 knockout [KO]) and the correspond-ing wild-type (WT) control mice (22). These mice wereprovided by D. Collen (University of Leuven, Leuven,Belgium). Nine-week-old female WT and PAI-1 KO micereceived a subcutaneous implant with slow-release pel-lets containing either 1.5 mg of corticosterone or pla-cebo (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 ineach group were 11 and 6, respectively. The animalswere maintained in metabolic cages in the 12-h light/12-h dark cycle; they received food and water ad libi-tum. Four weeks after the first implant of corticoste-rone or placebo pellets, insulin and glucose tolerancetests were performed. Quantitative computed tomogra-phy (qCT) was used to measure bone mineral density(BMD) in the tibia. Mice (placebo- and corticosterone-treated groups) were starved for 6 h before euthani-zation; blood and tissue samples were collected fromthe dead mice. All experiments were performedaccording to the guidelines of the National Institutesof Health and as per the institutional rules put forthfor the use and care of laboratory animals at KinkiUniversity.

Metabolic ParametersThe 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 contentin liver and muscle were measured (25,26). The plasmaconcentration of uncarboxylated OCN (ucOCN) was mea-sured using Mouse Undercarboxylated Osteocalcin ELISAKit (MyBiosource, San Diego, CA). Glucose and insulin tol-erance tests were performed as previously described (25).

qCT AnalysisFor the qCT analysis of body fat composition, BMD, andmuscle volume, the mice were scanned using a LaThetaLCT-200 experimental animal CT system (Hitachi AlokaMedical, Tokyo, Japan).

Histological AnalysisThe tibia, muscle, and liver tissues were fixed in 4%paraformaldehyde for 16 h at 4°C, and the tibia was fur-ther fixed for 7 days in 80% ethanol. Muscle and livertissues were embedded in paraffin right away, and thetibia was embedded in paraffin after dehydration withformic acid. A paraffin block was sliced into 4-mm sec-tions. For visualization of osteoclasts, the slices werestained using a TRAP/ALP Staining Kit (Wako Pure In-dustries, Osaka, Japan). Immunostaining with alkalinephosphatase (ALP) was performed (27). TUNEL stainingwas performed to identify apoptotic cells, using In SituCell Death Detection Kit, Fluorescein (Roche Diagnostics,Tokyo, Japan). The numbers of osteoclasts and TUNEL-positive and ALP-positive cells were counted in selectedvisual fields under a microscope in a blinded evaluation(16,27).

Quantitative Real-Time PCRTotal RNA was extracted from the homogenized tissuesamples and the cultured cells using the RNeasy Mini Kit(Qiagen, Tokyo, Japan). Real-time PCR was performedusing StepOnePlus and the Fast SYBR Green PCR MasterMix (Life Technologies, Tokyo, Japan) as previouslydescribed (25). The primer sets are shown in Supplemen-tary Table 1. The mRNA levels in tissues of the mice andin the cultured cells were normalized to b-actin and glyc-eraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA,respectively.

Cell Culture3T3-L1 cells and C2C12 myoblasts were induced todifferentiate into adipocytes and myotubes, respectively(28,29). The differentiated C2C12, 3T3-L1, and HepG2 cellswere preincubated with human active PAI-1 (MolecularInnovations, Novi, MI) and dexamethasone (Sigma-AldrichJapan, Tokyo, Japan) in the serum-free DMEM for 24 h.The cells were incubated with 100 nmol/L insulin for15 min. Protein levels of phosphorylated Akt (Ser473) andtotal Akt were measured by Western blotting (30). Theexpressions of gluconeogenesis-related genes in hepatocyteswere measured; HepG2 cells were incubated with 20 nmol/Lhuman active PAI-1 and 100 nmol/L dexamethasone for

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24 h in the presence of as well as without 1 nmol/L insulin.Insulin-stimulated 2-deoxyglucose (2DG) uptake in HepG2,C2C12, and 3T3-L1 cells was assessed using 2DG UptakeMeasurement Kit (Cosmo Bio, Tokyo, Japan).

Cell Proliferation and Apoptosis in Primary Osteoblastsand MC3T3E1 CellsPrimary osteoblastic cells were obtained from female WTand PAI-1 KO mouse calvaria (27). Cell proliferation, ap-optosis, and cell death rates of primary osteoblasts andMC3T3E1 cells were analyzed using the BrdU Cell Pro-liferation Assay Kit (Exalpha Biologicals, Shirley, MA); InSitu Cell Death Detection Kit, Fluorescein; and trypanblue staining, respectively.

Statistical AnalysisAll data were expressed as mean 6 SEM. Two-way ANOVAand two-way repeated-measures ANOVA were used to com-pare the effects of the placebo and corticosterone pellet onboth mouse genotypes (PAI-1 WT and PAI-1 KO) for non-repeated and repeated measures, respectively. When signif-icant differences were observed, individual means werecompared using Tukey post hoc test. For the other simplecomparisons between two groups, the unpaired Student ttest was used. Statistical values at P, 0.05 were consideredsignificant. All calculations were performed in the StatViewsoftware, version 5.0 (SAS Institute).

RESULTS

Effects of GC on Adiposity and Levels of CirculatingPAI-1Corticosterone treatment increased body weight in femaleand male WT mice, compared with placebo-treated WTmice (Fig. 1A and B). The qCT analysis showed that thepercentage of fat mass, visceral fat, and subcutaneousfat mass were markedly increased by corticosterone inboth sexes of WT mice, compared with the correspondingcontrol WT mice (Fig. 1C–F). The PAI-1 deficiency did notaffect the body weight gain, percentage of fat mass, vis-ceral fat, and subcutaneous fat mass either in placebo- orcorticosterone-treated female and male mice (Fig. 1A–F).The levels of plasma PAI-1 were markedly higher in bothsexes of corticosterone-treated WT mice, compared withthe corresponding control WT mice (Fig. 1G and H). Thelevels of PAI-1 mRNA were markedly and significantlyelevated in adipose and muscle tissues but significantlydecreased in the liver and spleen of corticosterone-treatedWT mice compared with placebo-treated WT mice.There were no differences in the levels of PAI-1mRNA in tibia, heart, lung, and kidney between theplacebo- and corticosterone-treated WT mice (Fig. 1I).

Effects of the PAI-1 Deficiency in GC-InducedAbnormal Glucose and Lipid MetabolismThe PAI-1 deficiency significantly suppressed the fastinglevels of blood glucose and the fasting plasma levels ofinsulin that were elevated by corticosterone in both sexesof mice (Fig. 2A–D).

The decrease in blood glucose levels after intraperitonealinsulin injection was attenuated in corticosterone-treated

WT mice; the PAI-1 deficiency significantly reversed theinsulin intolerance of corticosterone-treated WT mice(Fig. 2E and F), although the reversion of blood glucoselevels by the PAI-1 deficiency was not significant 90 and120 min after the insulin injection in male mice. Thelevels of blood glucose after intraperitoneal glucose injec-tion were markedly elevated in corticosterone-treated WTmice compared with placebo-treated WT mice (Fig. 2Gand H). The PAI-1 deficiency significantly suppressedthe elevation of blood glucose levels in corticosterone-treated mice 60, 90, and 120 min after the glucose in-jection (Fig. 2G and H). Moreover, the PAI-1 deficiencysignificantly suppressed the levels of plasma insulin in themice 30 min after the glucose injection (Fig. 2I and J).

Active PAI-1 significantly suppressed Akt phosphory-lation and glucose uptake induced by insulin in HepG2cells (hepatocytes), although active PAI-1 did not affectthem in differentiated C2C12 cells (myotubes) anddifferentiated 3T3-L1 cells (adipocytes) (Fig. 3A–F). Onthe other hand, dexamethasone suppressed Akt phos-phorylation and glucose uptake induced by insulin inHepG2, differentiated C2C12, and differentiated 3T3-L1cells (Fig. 3A–F), whereas the effects of both dexametha-sone and PAI-1 treatment were not additive in HepG2cells (Fig. 3A and D). Moreover, active PAI-1 and dexa-methasone did not affect the mRNA levels of key enzymesof gluconeogenesis such as glucose-6-phosphatase (G6Pase)and PEPCK in the presence of insulin in HepG2 cells, al-though insulin suppressed them in the absence of PAI-1and dexamethasone (Fig. 3G and H).

Corticosterone markedly increased the levels of plasmaTG and T-Chol in female and male WT mice compared withplacebo-treated WT mice, whereas the PAI-1 deficiency didnot affect the levels of these plasma lipids in corticosterone-treated mice (Fig. 4A–D). The PAI-1 deficiency did not seemto affect corticosterone-induced lipid accumulation in theadipose tissue and liver of the mice in histological analysis(Supplementary Fig. 1A and B). The PAI-1 deficiency did notaffect hepatic and muscular TG contents that were elevatedby corticosterone (Supplementary Fig. 1D and E).

Effects of the PAI-1 Deficiency on GC-Induced BoneLossThe qCT analysis revealed that corticosterone treatmentsignificantly reduced BMD, bone volume fraction (trabec-ular bone volume/total bone volume [BV/TV]), and trabec-ular area of the tibia in female and male WT mice,compared with placebo-treated WT mice (Fig. 5A–H, O,and P), although it did not affect cortical thickness, corticalarea, and total cross-sectional area in the tibia of WT mice(Fig. 5I–N). In contrast, the PAI-1 deficiency blunted thecorticosterone-induced decrease in BMD, BV/TV, and tra-becular area in both sexes of mice (Fig. 5A–H, O, and P).

Effects of the PAI-1 Deficiency in GC-InducedAbnormal Bone MetabolismCorticosterone treatment markedly suppressed the levelsof osteogenic genes, such as Runx2, Osterix, Alp, Ocn, and

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type 1 collagen (Col1) mRNA in tibia as well as the levelsof plasma OCN and ucOCN in WT mice, compared withthe placebo-treated WT mice (Fig. 6A–C). The PAI-1 de-ficiency did not affect these osteogenic markers when theywere downregulated by corticosterone in mice (Fig. 6A–C).In contrast, immunohistochemical analysis revealed thatthe PAI-1 deficiency attenuated the corticosterone-induced decrease in the number of ALP-positive osteoblasticcells on the bone surface of tibia in mice (Fig. 6D). Whencompared with the tibia of placebo-treated WT mice,

corticosterone suppressed the levels of receptor activatorof nuclear factor-кB ligand (RANKL) mRNA, osteoprote-gerin (OPG) mRNA, the RANKL/OPG ratio, and the num-ber of tartrate-resistant acid phosphatase (TRAP)–positive osteoclasts in the tibia of WT mice (Fig. 6E). Incontrast, corticosterone did not affect the levels of plasmaCTX, a marker for bone resorption, in WT mice (Fig. 6E).The PAI-1 deficiency did not affect these bone resorptionmarkers in either placebo- or corticosterone-treated mice(Fig. 6E).

Figure 1—Effects of GC on the levels of circulating PAI-1 and on PAI-1 expression in mice. The growth curve during experiments (A and B),the percentage of fat mass (C and D), and visceral and subcutaneous fat mass (E and F ) in placebo-treated (Cont) WT, corticosterone-treated (CS) WT, Cont PAI-1 KO, and CS PAI-1 KO female and male mice. ##P < 0.01 vs. Cont WT mice; **P < 0.01 vs. Cont KO mice (n =11 and 6 in each group of female and male mice, respectively). The levels of plasma PAI-1 (G and H) in both sexes of Cont and CSWT 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 areshown relative to b-actin mRNA values (mean 6 SEM). Two-way repeated-measures ANOVA and two-way ANOVA (2WA) were used forgrowth curve (A and B) and for fat mass (C–F), respectively, to compare the drug effects (d) of placebo and the corticosterone pellets (Contand CS) on both the genotypes of mice (PAI-1 WT and PAI-1 KO). When significant differences were observed among individual means,post hoc analysis was performed using Tukey test. For assessment of body fat composition using a qCT scan, contiguous 2-mm sliceimages between L1 and L5 were used for quantitative assessment in the LaTheta software (version 3.40). Visceral fat and subcutaneous fatwere identified, and the total fat content, visceral fat weight, and subcutaneous fat weight were calculated from all slice images and wereevaluated quantitatively. The percentage of fat mass was calculated by dividing total fat mass by total body mass.

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Role of PAI-1 in the Apoptosis and Proliferation ofOsteoblastsCorticosterone treatment increased the number of TUNEL-positive cells on the bone surface of tibia of WT mice,compared with placebo-treated mice (Fig. 7A). The PAI-1deficiency significantly blunted the corticosterone-induced increase in the number of TUNEL-positive cellson the bone surface of the tibia of the mice (Fig. 7A).Moreover, an in vitro experiment showed that active

PAI-1 significantly increased the number of trypan blue–stained and TUNEL-positive MC3T3E1 cells and primaryosteoblasts obtained from mouse calvaria (Fig. 7B–E). Incontrast, active PAI-1 did not affect BrdU incorporationinto MC3T3E1 cells and into primary osteoblasts obtainedfrom mouse calvaria (Fig. 7F and G), indicating thatexogenous PAI-1 did not affect the proliferation ofosteoblasts in vitro. Moreover, the PAI-1 deficiency didnot affect the proliferation impaired by dexamethasone

Figure 2—Effects of the PAI-1 deficiency on GC-induced abnormal glucose metabolism in female mice. The fasting levels of blood glucose(A and B) and plasma insulin (C and D) in placebo-treated (Cont) WT, corticosterone-treated (CS) WT, Cont PAI-1 KO, and CS PAI-1 KOfemale and male mice. The results are presented as mean 6 SEM. *P < 0.05, **P < 0.01 (n = 11 and 6 in each group of female and malemice, respectively). Responses of blood glucose to a single intraperitoneal injection of insulin (E and 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 andmale mice, respectively). Responses of blood glucose levels (G and H) to a single intraperitoneal injection of glucose in Cont WT (opencircles), 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 ineach group of female and male mice, respectively). Responses of plasma insulin (I and J) to a single intraperitoneal injection of glucose inCont 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 6 SEM. *P < 0.05 and **P < 0.01 vs.CS WT mice. Two-way ANOVA (2WA) and two-way repeated-measures ANOVA were used for A–D and E–J, respectively, to compare thedrug effects (d) of a placebo and a corticosterone pellet (Cont and CS) on the mice of two genotypes (g) (PAI-1 WT and PAI-1 KO). Whensignificant differences were observed, individual means of the bars were compared using Tukey post hoc test.

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in primary osteoblasts obtained from mouse calvaria(Fig. 7H).

Effects of the GC-Induced PAI-1 Deficiency on MuscleWastingThe qCT analysis revealed that corticosterone treatmentsignificantly reduced muscle mass in the whole body ofWT mice compared with placebo-treated mice (Fig. 8A andB). Corticosterone significantly decreased the tissueweight of the gastrocnemius muscle, but not of the soleusmuscle, in WT mice (Fig. 8C and D). This result was sug-gestive of the predominant involvement of type II muscle

fibers in the GC-induced muscle wasting. The PAI-1 de-ficiency attenuated the corticosterone-induced decrease intissue weight of the gastrocnemius muscle in both sexesof mice (Fig. 8C and D). Moreover, the PAI-1 deficiencyattenuated the corticosterone-induced decrease in the cross-sectional area of myofibers in the gastrocnemius muscles infemale mice (Fig. 8E and Supplementary Fig. 1C). Corti-costerone significantly decreased the levels of muscledifferentiation–related genes, such as MyoD mRNA, inthe gastrocnemius muscle tissues of WT female mice. Incontrast, corticosterone did not affect the mRNA levels of

Figure 3—The influence of PAI-1 and dexamethasone (Dex) on insulin signaling in hepatocytes, myocytes, and adipocytes in vitro.Phosphorylation of Akt (p-Akt), stimulated by insulin treatment (100 nmol/L) for 15 min with or without active PAI-1 (20 nmol/L) and Dex(100 nmol/L) treatment for 24 h in HepG2 cells (A), differentiated C2C12 cells (B), and differentiated 3T3-L1 cells (C). 2DG uptake (theconcentration of 2-deoxyglucose-6-phosphate [2DG6P]) stimulated by insulin treatment (1 mmol/L) for 20 min with or without active PAI-1(20 nmol/L) and Dex (1 mmol/L) treatment for 24 h in HepG2 cells (D), differentiated C2C12 cells (E), and differentiated 3T3-L1 cells (F ). Theresults are presented as mean 6 SEM of three experiments. The mRNA levels of G6Pase (G) and PEPCK (H) in HepG2 cells incubatedwith or without active PAI-1 (20 nmol/L) and Dex (100 nmol/L) with and without insulin (1 nmol/L) for 24 h. The results are presented asmean 6 SEM of six experiments. *P < 0.05; **P < 0.01. Tukey post hoc test was used. Cont, vehicle treated.

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myogenin and myosin heavy chain (MHC) in the gastrocne-mius muscle tissue of female mice (Fig. 8F). The PAI-1 de-ficiency significantly attenuated the corticosterone-induceddecrease in the levels of MyoD mRNA in mice (Fig. 8F),suggesting that PAI-1 was involved in muscle differentia-tion suppressed by GCs at an early differentiation stage.

DISCUSSION

GC treatment simultaneously induces multiple metabolicdisorders in an individual (4). Therefore, it is importantto identify a common therapeutic target for GC-inducedadverse effects. We found that PAI-1 levels are associatedwith major GC-induced adverse metabolic effects in mice,for example, insulin resistance, osteoporosis, and musclewasting.

Studies suggest that PAI-1 is linked to insulin re-sistance as well as metabolic abnormalities (15,31). Werecently reported that PAI-1 deficiency ameliorates insu-lin resistance and hyperlipidemia in obese female mice(25). For the first time, we demonstrated that PAI-1 de-ficiency reduces insulin resistance and glucose intolerancebut 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 signalingin 3T3-L1 adipocytes, L6 myotubes, and HepG2 hepato-cytes (30,32,33). In this study, dexamethasone inhibitedthe phosphorylation of Akt and the glucose uptake in-duced by insulin in 3T3-L1 adipocytes, HepG2 hepato-cytes, and C2C12 myotubes in vitro. Nevertheless,incubation with exogenous PAI-1 suppressed insulin sig-naling in HepG2 hepatocytes but not in 3T3-L1 adipo-cytes and C2C12 myotubes. Moreover, active PAI-1seemed to blunt the levels of key enzymes of gluconeo-genesis suppressed by insulin in HepG2 cells. Thus, PAI-1participates in GC-induced insulin resistance by influenc-ing hepatocytes.

Seki et al. (34) reported that dexamethasone increasesPAI-1 production in 3T3-L1 adipocytes. Our study showedthat corticosterone treatment elevates the levels of PAI-1mRNA in adipose and muscle tissues of mice. The extentof PAI-1 upregulation was much higher in adipose tissuesthan in the muscle tissues. This suggests that circulatingGC-induced PAI-1 produced in adipose and muscle tissuesmight cause GC-induced whole-body insulin resistance byimpairing insulin signaling in the liver via the blood-stream but not in adipose and muscle tissues. The targetorgans for inhibition of insulin signaling by GC and PAI-1seem to be different. PAI-1 circulates in plasma as a com-plex with vitronectin, an extracellular matrix glycoprotein(35), stabilizing active conformation of PAI-1. López-Alemany et al. (36) showed in vitro that PAI-1 inhibitsinsulin signaling by competing with integrin avb3 forvitronectin binding. Possibly PAI-1 deficiency amelioratedGC-induced insulin resistance via enhancement of the in-teraction with vitronectin and avb3 integrin in the liver.PAI-1 suppression of insulin signaling in hepatocytes, butnot in myotubes and adipocytes, is still unknown. Furtherstudies are needed to clarify the molecular mechanismunderlying PAI-1 effects on hepatocytes and differentPAI-1 tissue sensitivities.

Recent studies suggest that osteoblast-derived OCN,a bone matrix protein, is a potent regulator of glucosemetabolism, acting by modulating the insulin release andperipheral insulin sensitivity (37,38). Brennan-Speranzaet al. (23) showed that bone-derived OCN release, whichis impaired by GC, is involved in GC-induced abnormali-ties in glucose/lipid metabolism. Our study demonstratedthat the PAI-1 deficiency does not affect levels of carbox-ylated and uncarboxylated plasma OCN when suppressedby GC in mice; this deficiency significantly reducesGC-induced insulin resistance. This suggests that the in-volvement of PAI-1 in GC-induced insulin resistance isindependent of OCN in mice.

Figure 4—Effects of the PAI-1 deficiency on hyperlipidemia induced by GC in mice. The levels of plasma TG (A and B) and T-Chol (C and D)in placebo-treated (Cont) WT, corticosterone-treated (CS) WT, Cont PAI-1 KO, and CS PAI-1 KO female and male mice. The results areshown as mean 6 SEM (n = 11 and 6 in each group of female and male mice, respectively). Two-way ANOVA (2WA) was used to comparethe drug effects (d) of the placebo and corticosterone pellet (Cont and CS) on the mice of two genotypes (PAI-1 WT and PAI-1 KO). Whensignificant differences were observed, individual means of the bars were compared using Tukey post hoc test.

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Figure 5—Effects of the PAI-1 deficiency on bone loss induced by GC treatment in mice. BMD values in total (A and B), trabecular (C andD), and cortical bones (E and F ); bone volume fraction (BV/TV) (G and H); cortical thickness (I and J); cortical area (K and L); total cross-sectional area (M and N ); and trabecular area (O and P) of the tibia in placebo-treated (Cont) WT, corticosterone-treated (CS) WT, ContPAI-1 KO, and CS PAI-1 KO female and male mice. The results are presented as mean 6 SEM. *P < 0.05; **P < 0.01 (n = 11 and 6 in eachgroup of female and male mice, respectively). Two-way ANOVA (2WA) was used to compare the drug effects (d) of the placebo andcorticosterone pellet (Cont and CS) on the mice of two genotypes (g) (PAI-1 WT and PAI-1 KO). When significant differences wereobserved, individual means of the bars were compared using Tukey 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 mm distal to the end of theproximal growth plate over 1.5 mm toward the diaphysis. For assessment of cortical BMD, thickness, and area, ROIs were defined as2.0-mm segments of the mid-diaphysis tibia. For assessment of total BMD and total cross-sectional area, ROIs were defined as 9,600-mmsegments (100 slices) from the distal end of the proximal growth plate of the tibia. The bone volume fraction was calculated as the ratio ofBV (mm3) to TV (mm3). Parameters used for the CT scans were as follows: tube voltage, 50 kVp; tube current, 500 mA; integration time,3.6 ms; axial field of view, 48 mm; and voxel size of 48 3 96 mm with a slice thickness of 96 mm. Bone parameters were analyzed using theLaTheta software (version 3.40).

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This study is the first to report that PAI-1 deficiencyameliorates GC-induced bone loss and decrease in osteo-blast numbers in mice. Long-term GC treatment causesbone loss mainly due to the impaired osteoblastic boneformation (4). GCs decrease the number of osteoblastsbecause of the impaired osteoblast differentiation andproliferation as well as enhanced apoptosis of osteoblasts,resulting in impairment of bone formation (4). Accordingto our data, corticosterone markedly suppressed levels ofosteogenic genes in the tibia of mice, indicating that GCsimpair osteoblast differentiation. The PAI-1 deficiency didnot affect the osteoblast differentiation suppressed byGC. This suggests that PAI-1 is not linked to the impairedGC-induced osteoblast differentiation. Also, either endogenous

or exogenous PAI-1 does not affect the proliferation ofosteoblasts in vitro. In contrast, the PAI-1 deficiency sup-pressed corticosterone-induced apoptosis in the tibia ofmice. Moreover, active PAI-1 induced apoptosis in primaryosteoblasts and MC3T3E1 cells. Overall, this indicates thatGCs induce osteopenia through PAI-1 presumably via theenhancement of apoptosis in osteoblasts.

The enhancement of osteoclastic bone resorption thatis induced by excess GC is also associated with GC-induced bone loss (4). During the initial stage of high-dose GC therapy, a rapid but transient increase in boneresorption caused by increase in osteoclast number andactivity can be observed in humans and in animal models(39,40). Nonetheless, corticosterone decreased the number

Figure 6—Effects of the PAI-1 deficiency on abnormal bone metabolism induced by GC treatment in female mice. The levels of Runx2,Osterix, Alp, Ocn, and Col1 mRNA in the tibia (A), plasma OCN (B), and plasma ucOCN (C ) in placebo-treated (Cont) WT (n = 5),corticosterone-treated (CS) WT (n = 5), Cont PAI-1 KO (n = 5), and CS PAI-1 KO (n = 5) female mice. The number of ALP-positive cellson 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 ofRANKL and OPG mRNA in tibia and RANKL/OPG ratio, plasma cross-linked CTX, and the number of TRAP-positive multinucleated cells inthe 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 asmean 6 SEM. *P < 0.05. The results of mRNA expression are shown relative to b-actin mRNA values. Two-way ANOVA (2WA) was usedto compare the drug effects (d) of the placebo and corticosterone pellet (Cont and CS) on the mice of two genotypes (g) (PAI-1 WT andPAI-1 KO). When significant differences were observed, individual means of the bars were compared by means of Tukey post hoc test.

2202 PAI-1 and Glucocorticoid-Induced Adverse Effects Diabetes Volume 64, June 2015

of osteoclasts, the levels of RANKL mRNA, and theRANKL/OPG ratio in the tibia of mice. It is suggestedthat excess GC for a prolonged period can downregulateosteoclast numbers and function (41). Furthermore, GCsuppresses the formation of osteoclast precursors (41,42).Therefore, we can speculate that the suppression of oste-oclast formation by GC in this study might partly be dueto the dose and/or duration of GC administration. ThePAI-1 deficiency did not affect either osteoclast numbersor the levels of tibia RANKL and OPG mRNA (suppressedby the GC), suggesting that PAI-1 is not involved in thechanges of GC-induced bone resorption.

Clinical evidence suggests that GCs negatively affectboth muscle and the bone; this phenomenon may lead tomuscle wasting as well as osteoporosis (43–45). Our datasuggest that PAI-1 deficiency blunts the decrease in musclemass and the GC-induced changes in muscle phenotypes inmice, indicating that PAI-1 is also involved in GC-inducedmuscle wasting in mice. Because GCs significantly enhanced

endogenous PAI-1 levels in muscle tissues, GCs might in-duce muscle wasting in mice through both muscle’s en-dogenous PAI-1 and circulating PAI-1.

GCs directly enhance transactivation of PAI-1 throughbinding of a GC receptor to a GC response element in PAI-1’spromoter (46). Several studies showed that GCs enhance PAI-1expression in various cell types (34,47). However, thesource tissue of the PAI-1 production enhanced by GCin vivo has not been identified. In the current study, GCsignificantly decreased the levels of PAI-1 mRNA in theliver and spleen, although the GC elevated PAI-1 mRNAlevels in adipose and muscle tissues in mice. Oishi et al.(48) reported that serum GC concentrations are corre-lated with PAI-1 expression in adipose tissues but notin the liver of mice. In the same study, it was shownthat GC-induced adipose tissue–derived PAI-1 possiblyhad negative effects on the GC-induced liver-derived PAI-1production. Additionally, our previous study showedthat the diabetic state increases PAI-1 expression in the

Figure 7—The influence of PAI-1 on apoptosis and proliferation of osteoblasts. The number of TUNEL-positive cells on the bone surface inthe tibia of placebo-treated (Cont) WT (n = 5), corticosterone-treated (CS) WT (n = 5), Cont PAI-1 KO (n = 5), and CS PAI-1 KO (n = 5) femalemice (A). Two-way ANOVA (2WA) was used to compare the drug effects (d) of the placebo and corticosterone pellet (Cont and CS) on themice of two genotypes (g) (PAI-1 WT and PAI-1 KO). When significant differences were observed, individual means of the bars werecompared using Tukey post hoc test. The cell death rate in primary osteoblasts (OBs) from female mouse calvaria (B) and MC3T3E1 cells(C ) incubated with or without active PAI-1 (20 nmol/L) for 24 h. Cell death was identified by trypan blue staining. The number of apoptoticprimary OBs from female mouse calvaria (D) and MC3T3E1 (E) cells incubated with or without active PAI-1 (20 nmol/L) for 24 h. Apoptoticcells were identified by TUNEL staining. The results are shown as mean 6 SEM of three experiments. *P < 0.05, **P < 0.01. UnpairedStudent t test was used. Proliferation of primary OBs from WT female mouse calvaria (F ) and MC3T3E1 cells (G) incubated with or withoutactive PAI-1 (20 nmol/L) for 24 h. Proliferation of primary OBs from female WT and PAI-1 KO mouse calvaria incubated with or withoutdexamethasone (Dex; 100 nmol/L) (H). Cell proliferation was assessed using a BrdU incorporation assay. The results are shown as mean 6SEM of three experiments. Two-way ANOVA (2WA) was used to compare the drug effects (d) of vehicle and Dex (Cont and Dex) on the miceof two genotypes (g) (PAI-1 WT and PAI-1 KO). When significant differences were observed, individual means of the bars were comparedusing Tukey post hoc test.

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liver of female mice (16). Thus, we can speculate thatthere are tissue-specific differences in the PAI-1 induc-tion by GC. The response of different tissue-specificfactors related to GC may negatively affect the PAI-1induction by GC, depending on an organ. Alternatively,the change in the metabolic state may modulate thePAI-1 expression in response to GC in different ways,depending on a tissue.

The reversion of blood glucose levels by the PAI-1deficiency was not significant at 90 and 120 min afterinsulin injection in male mice (Fig. 2F). The blood glucoselevels after insulin injection seemed to be higher (notsignificant) in PAI-1–deficient mice than in the WTmice, suggesting that slight sex differences might existin PAI-1 involvement in the pathogenesis of GC-inducedinsulin resistance in mice. We previously reported thatPAI-1 is involved in the pathogenesis of type 1 diabeticosteoporosis 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 theprevalence of metabolic abnormality and osteoporosis(49,50). This suggests that a protein linked to a sex chro-mosome might be responsible for the sex differences ob-served in the current study. Further studies are needed toclarify the above issues.

PAI-1 is considered as a humoral factor that affectssystemic metabolism. Our previous study suggested thata type 1 diabetic state increases PAI-1 expression in theliver, resulting in increased circulating PAI-1 levels infemale mice (16). Upregulation of circulating PAI-1impairs osteoblast differentiation and mineralizationand promotes adipogenesis in bone tissues; the latter ef-fect leads to diabetic osteoporosis. Moreover, PAI-1 isinvolved in obesity-induced insulin resistance and hyper-lipidemia but not bone loss in female mice, although therole of PAI-1 in bone metabolism in the type 2 diabetic stateremains completely unknown at present (25). Our studysuggested that GC increases PAI-1 production mainly in

Figure 8—Effects of the PAI-1 deficiency on muscle wasting induced by GC treatment in mice. Total muscle mass (A and B) in placebo-treated (Cont) WT, corticosterone-treated (CS) WT, Cont PAI-1 KO, and CS PAI-1 KO female and male mice. The results are presented asmean 6 SEM. **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 and 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 ofMyoD,myogenin, andMHC (F) in the gastrocnemius muscle in Cont WT, CS WT, Cont PAI-1KO, and CS PAI-1 KO female mice. For assessment of the cross-sectional area of a myofiber, a minimum of 1,000 myofibers per muscle wereanalyzed on a hematoxylin and eosin–stained gastrocnemius muscle slice. The results are presented as mean6 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 placeboand corticosterone pellet (Cont and CS) on the mice of two genotypes (g) (PAI-1 WT and PAI-1 KO). When significant differences wereobserved, individual means of the bars were compared using Tukey 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). G: Theproposed hypothesis for the role of PAI-1 in the pathogenesis of insulin resistance, bone loss, and muscle wasting induced by GC in miceis that GC increases PAI-1 expression in adipose tissue, resulting in elevation of circulating PAI-1 levels in mice. An elevated level ofcirculating PAI-1 induces insulin resistance in the liver, bone loss due to the increased apoptosis of osteoblasts, and muscle wasting in mice.

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adipose tissues, thereby leading to increased blood PAI-1levels. The upregulation of circulating PAI-1 may lead toosteoporosis as well as insulin resistance and muscle wastinginduced by GC (Fig. 8G). Thus, PAI-1 possibly plays an im-portant role as a humoral factor to regulate various meta-bolic states in the presence of excess GC as well as indiabetes.

In conclusion, for the first time, we demonstrated thatPAI-1 is involved in the metabolic adverse effects of GCtreatment, for example, in insulin resistance, bone loss, andmuscle wasting in mice. PAI-1 may be a novel therapeutictarget that can help to decrease the risk of GC-inducedadverse outcomes. PAI-1 may serve as a diagnostic markerof GC-induced diabetes, osteoporosis, and muscle wasting.

Funding. This study was supported by grants-in-aid 26860152 and24590289 from the Ministry of Education, Culture, Sports, Science and Tech-nology of Japan (to Y.T. and H.K., respectively) and grants from the JapanOsteoporosis Foundation, the Takeda Science Foundation, and Kinki University.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. Y.T. researched data, contributed to the discus-sion, and wrote, reviewed, and edited the manuscript. N.K., M.Y., K.Oka., K.Oku.,Y.C., and O.M. contributed to the discussion and reviewed and edited the man-uscript. H.K. contributed to the discussion and wrote, reviewed, and edited themanuscript. H.K. is the guarantor of this work and, as such, had full access to allthe data in the study and takes responsibility for the integrity of the data and theaccuracy of the data analysis.

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