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Klotho Protein Promotes Adipocyte Differentiation

Department of Geriatric Medicine (Y.C., H.R., K.I., M.I., Y.M., J.O., I.K., T.O.), Osaka University Graduate School of Medicine, Osaka 565-0871, Japan; and Department of General Medicine (K.I.), Osaka University Hospital, Osaka 565-0871, Japan

Address all correspondence and requests for reprints to: Hiromi Rakugi, M.D., Ph.D., Department of Geriatric Medicine, Osaka University Graduate School of Medicine, 2-2, Yamada-oka, Number B6, Suita, Osaka 565-0871, Japan. E-mail: rakugi{at}geriat.med.osaka-u.ac.jp.

	Abstract

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Mice with homozygous disruption of the klotho exhibit multiple age-related disorders and have barely detectable amounts of white adipose tissue. Although klotho expression in cultured adipocytes has been reported, little is known about its function in adipocytes. In the present study, we investigated the role of klotho on adipocyte differentiation. Adipocyte differentiation was induced by incubation of confluent 3T3-L1 cells with insulin, dexamethasone, and 1-methyl-3-isobutyl-xanthin. Klotho-siRNA and expression vector were produced for klotho suppression and overexpression, respectively. Klotho protein was purified for determination of the hormonal effect of klotho. Klotho mRNA and protein expression increased up to the 3rd d of differentiation. A peroxisome proliferator-activated receptor- agonist increased klotho expression during the early period of adipocyte differentiation. The mRNA expression of adipocyte differentiation markers, such as CCAAT/enhancer-binding protein (C/EBP), C/EBPß, C/EBP, peroxisome proliferator-activated receptor-, and fatty acid binding protein 4, was decreased by klotho suppression, and increased 1.9- to 3.8-fold by klotho overexpression. The results of Oil Red O staining also suggested that klotho overexpression promoted adipocyte differentiation. Klotho protein stimulation resulted in a 2.4- to 4.6-fold increase in mRNA expression of differentiation markers compared with control, and the time course depended on adipocyte induction status. Western blot analysis showed that protein levels of C/EBP and C/EBP were increased by Klotho protein stimulation. These results suggest that klotho works as a hormonal factor to promote adipocyte differentiation in the early days, during the period of transient proliferation in the differentiation process, and that klotho may play an essential role in adipocyte differentiation.

	Introduction

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MICE WITH HOMOZYGOUS disruption of the klotho gene exhibit multiple age-related disorders observed in humans, including skin atrophy, ectopic calcification, osteoporosis, and atherosclerosis (1, 2). On the contrary, overexpression of klotho in mice extends lifespan (3). Klotho gene is expressed principally in the distal tubules of the kidney, the choroid plexus in the brain, the parathyroid, the testis, and the ovary (1, 4, 5, 6). Although klotho is expressed in limited tissues, we and others (3, 7) have reported that Klotho protein functions as a humoral factor.

The function of klotho is not well known; some reports, however, have identified a relationship between insulin/glucose metabolism and klotho in vivo and in vitro (3, 8, 9, 10). In klotho-disrupted mice, blood glucose was reduced, and insulin sensitivity was elevated (8). In Otsuka Long-Evans Tokushima Fatty rats, which exhibit hypertension, obesity, and glucose metabolism abnormalities, klotho expression is decreased, and the peroxisome proliferator-activated receptor (PPAR)- agonist, troglitazone, increased klotho gene expression in the kidney (9).

Moreover, in 3T3-L1 cells (mouse fibroblasts), the membrane form of the Klotho protein showed increased expression during differentiation into adipocytes, and the thyroid hormone triiodothyronine increased klotho expression in differentiated adipocytes (10). Adipocytes are known to contribute to the development of vascular diseases closely related to aging disorders via production of adipocytokines. Of interest, mice with disruption of the klotho gene have barely detectable amounts of white adipose tissue (11) and klotho suppresses insulin and IGF-I signaling (3), which greatly affects adipocytes differentiation (12). These issues led us to investigate the role of klotho in the differentiation of adipocytes.

It has been reported that the differentiation of adipose precursor cells can be divided into early and late events, a phase of proliferation and a phase of differentiation (13, 14). A number of genes have been shown to be differentially expressed during adipocyte differentiation. Some of them are involved in lipid synthesis and storage, and others, such as novel transcription factors, are induced during differentiation.

In the present study, we investigated klotho expression during the differentiation induced by several stimulations. Furthermore, to elucidate the role of klotho in the differentiation of adipocytes, we investigated the effects of suppression and overexpression of klotho gene on adipocyte differentiation.

	Materials and Methods

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Cell culture
Mouse 3T3-L1 cells (American Type Culture Collection, Manassas, VA) in six-well plates were proliferated in DMEM (Life Technologies, Inc.-BRL, Gaithersburg, MD) that contained 10% fetal bovine serum (FBS) at 37 C in 5% CO₂. After 3T3-L1 cells were kept confluent for 2 d, they were incubated in induction medium (d 0, 0 h) containing 1 µM dexamethasone (Dex) (Sigma-Aldrich), 0.5 mM 1-methyl-3-isobutyl-xanthin (IBMX) (Sigma-Aldrich), and 5 µg/ml insulin (Sigma-Aldrich) in DMEM with 10% FCS for 48 h of adipocyte differentiation. Thereafter, the medium in the cell culture plates was replaced with DMEM with 10% FBS. The medium was changed every other day (15).

To assess the effect of PPAR- agonist on klotho expression, we used 10 µM pioglitazone (generous gift from Takeda Chemical Industries, Osaka, Japan) instead of induction medium after 2 d of confluency.

Klotho gene suppression by small interfering RNA (siRNA)
Klotho-siRNA plasmid was constructed using the Gene Suppressor System (IMGENEX Corp., San Diego, CA) for knockdown of the klotho gene in vitro, as previously described (16). Klotho-siRNA plasmid was transfected into 3T3-L1 cells using the Trans-IT-3T3 Transfection Kit (Mirus Corporation, Madison, WI), and the siRNA duplex was then synthesized within the cells. The sequence of klotho-specific siRNA used in the present study was 5'-GCGACTACCCAGAGAGTAT-3'. After klotho-siRNA plasmid was transfected into 3T3-L1 cells, cells were grown to confluency. After 2 d confluency, 3T3-L1 cells were incubated with insulin, Dex, and IBMX for adipocyte induction. We used control-siRNA (gene suppressor system, 5'-TCAGTCACGTTAATGGTCGTT-3') for control experiment.

Plasmid construction
Plasmid pCAGGS-klotho was constructed by inserting the complete mouse membrane form klotho of cDNA (generous gift from Professor Yo-ichi Nabeshima, Department of Pathology and Tumor Biology, Kyoto University Graduate School of Medicine, Kyoto, Japan) (1) into the EcoRI site between the cytomegalovirus immediate early enhancer chicken ß-actin hybrid promoter and a 3'-flanking sequence of the rabbit ß-globin gene of the pCAGGS expression vector (generous gift from Professor Jun-ichi Miyazaki, Division of Stem Cell Regulation, Osaka University Graduate School of Medicine, Osaka, Japan). Plasmids were amplified in Escherichia coli DH5 cells, extracted using alkaline lysis methods, and purified using a QIAGEN Endo-free Plasmid Maxi Kit (QIAGEN K.K., Tokyo, Japan). Finally, the plasmids were dissolved in buffer (10 mM Tris-HCl, 1 mM EDTA). The quantity and quality of the purified plasmid DNA were assessed by determining optical density at 260 and 280 nm and also by agarose gel electrophoresis (17).

Transfection of plasmid DNA
When 3T3-L1 cells reached subconfluency, they were transfected with 1 µg/well of either pCAGGS for control or pCAGGS-klotho for klotho overexpression for 6 h in serum-free medium using Lipofectamine Plus 2000 (Life Technologies, Inc.-BRL), as described previously (17, 18). Thereafter, the medium was changed to DMEM with 10% FBS.

Quantification of klotho, CCAAT/enhancer-binding protein (C/EBP) , ß, and , PPAR-, fatty acid binding protein 4 (aP2), adiponectin, and leptin gene expression
Total RNA was extracted from the cells using the SV Total RNA Isolation kit (Promega, Madison, WI). To quantify mRNA expression of klotho, C/EBP, C/EBPß, and C/EBP, PPAR-, aP2, adiponectin, and leptin, the TaqMan-PCR (Applied Biosystems, Foster City, CA) method was used with a PRISM 7900 HT (Applied Biosystems), which can detect RNA amounts in real time using a fluorescent probe complementary to the RNA sequence. Expression levels of each gene were normalized to the control gene expression (18S rRNA gene).

Purification of recombinant mouse klotho
A 6xHis tag was inserted into the pCAGGS-klotho plasmid (3' end site of the klotho cDNA) for construction of the 6His-pCAGGS-klotho plasmid. The plasmid was transfected into COS-1 cells using Lipofectamine Plus 2000, and cells were grown for 48 h. The 6xHis-klotho protein was purified from cell lysate by affinity chromatography on nickel-nitrilotriacetic acid agarose gel (QIAGEN). Immunoblotting using anti-6xHis-tagged protein and antiklotho antibody (KM2076, generous gift from Kyowa Hakko Kogyo Co. Ltd., Tokyo, Japan) (19) demonstrated that the recombinant purified protein was coincident with the Klotho protein. At the same time as induction and every 2 d after induction, 2 nM Klotho protein was administered to the 3T3-L1 cells.

Western blot analysis
Cells were washed twice with PBS and harvested in a lysis buffer [1% SDS, 100 mM NaCl, 50 mM Tris-HCl (pH 8.0), and 20 mM EDTA]. The harvested cells were sonicated. After centrifugation, samples were boiled for 4 min. The samples were loaded onto a 12.5% SDS-PAGE gel and electroblotted onto nitrocellulose filters. Blots were blocked in 5% nonfat milk in PBS for 1 h with anti-Klotho rat monoclonal antibody (KM2076) (18, 19), anti-C/EBP, and anti-C/EBP (Santa Cruz Biotechnology, Santa Cruz, CA) and incubated with peroxidase-conjugated second antibodies for 1 h. To monitor equal loading of proteins, membranes were incubated with an -tubulin antibody (Calbiochem, La Jolla, CA). Immunoblots were developed using an ECL plus Western blotting detection system (GE Healthcare, Little Chalfont, Buckinghamshire, UK), as described previously (7, 17, 18).

Oil Red O staining
Cells were washed with PBS twice, stained with 0.5% Oil Red O solution (60% isopropanol) for 15 min at room temperature (20), washed, and removed with dye extraction solution. Stained Oil Red O was quantified by measuring the optical absorbance at 510 nm (21).

Quantification of GPDH activity
At d 8, cells were washed with PBS twice, enzyme extraction buffer was added, and cells were collected by scraping with a cell scraper. After centrifugation at 10,000 rpm for 5 min at 4 C, the glycerol-3-phosphate dehydrogenase (GPDH)-specific activity of supernatant was measured with a GPDH Activity Assay Kit (Takara, Kyoto, Japan). Values for GPDH activity were expressed as a proportion of control (100%).

Statistical analysis
Comparisons of data from various time points were performed using one-way ANOVA, followed by Duncan’s multiple range tests, using StatView (Abacus Concepts Inc., Berkeley, CA). All data are expressed as mean ± SEM. P < 0.05 was considered to indicate statistical significance.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Klotho expression in the 3T3-L1 cells during adipocyte differentiation
Klotho mRNA expression started increasing within 24 h, reaching up to 5.2-fold at d 3 (Fig. 1A). Expression decreased gradually after the d 3 peak. Klotho protein expression has shown the same time course as mRNA expression (Fig. 1B). These results show that klotho expression increased during the early period of adipocyte differentiation.

View larger version (17K): [in this window] [in a new window]   FIG. 1. The expression levels of klotho mRNA and protein during differentiation in 3T3-L1 cells. The relative levels of klotho mRNA expression (A) and protein expression (B) in 3T3-L1 cells during adipocyte differentiation. Experiments were performed four times in independent cultures. Data are expressed as relative to the average value at d 0 (3T3-L1 cells before induction of differentiation) which was set to 1, and expressed as mean ± SEM. *, P < 0.05 vs. 3T3-L1 cells before induction (0 d).

Changes in klotho mRNA expression by PPAR- agonist

View larger version (23K): [in this window] [in a new window]   FIG. 2. The expression levels of klotho mRNA by incubation with pioglitazone in confluent 3T3-L1 cells. The relative levels of klotho (A), C/EBP (B), and C/EBP (C) mRNA expression in 3T3-L1 cells by incubation with 10 µM pioglitazone (black circle) or normal induction (white circle). Experiments were performed four times in independent cultures. Data are expressed as relative to the average value at d 0 (3T3-L1 cells before induction of differentiation), which was set to 1 and expressed as mean ± SEM. *, P < 0.05 vs. 3T3-L1 cells before induction (0 d).

View larger version (28K): [in this window] [in a new window]   FIG. 3. The expression levels of adipocyte differentiation markers, C/EBP, C/EBPß, C/EBP, PPAR-, and aP2 mRNA, during adipocyte differentiation in 3T3-L1 cells by klotho suppression. 1, Suppression of klotho protein expression in 3T3-L1 cells by klotho-siRNA. The picture of Western blotting shows that klotho-siRNA, but not control-siRNA, specifically down-regulated klotho protein expression at 48 h after transfection, compared with nontransfection. Representative pictures are presented. 2, Relative level indicates C/EBP (A), C/EBPß (B), C/EBP (C), PPAR- (D), and aP2(E) mRNA expression in 3T3-L1 cells transfected with klotho-siRNA, control-siRNA, or without transfection. None, No transfection of siRNA; C, control-siRNA; K, klotho-siRNA. Experiments were performed four times in independent cultures. Each experiment consisted of three wells for control, three wells of control-siRNA transfection, and three wells for klotho-siRNA transfection. Data are expressed as relative to the average value of control, which was set to 1, and expressed as mean ± SEM. *, P < 0.05 vs. None; #, P < 0.05 vs. C.

View larger version (28K): [in this window] [in a new window]   FIG. 4. The expression levels of adipocyte differentiation markers, C/EBP, C/EBPß, C/EBP, PPAR-, and aP2 mRNA during adipocyte differentiation in 3T3-L1 cells by klotho overexpression. The relative levels of C/EBP (A), C/EBPß (B) and C/EBP (C), PPAR- (D), and aP2 (E) mRNA expression in 3T3-L1 cells transfected with pCAGGS-klotho (black circle), pCAGGS (white circle), or without transfection (triangle). Experiments were performed four times in independent cultures. Each experiment consisted of three wells for control, three wells for pCAGGS transfection, and three wells for pCAGGS-klotho transfection. Data are expressed as relative to the average value at d 0 (3T3-L1 cells before induction of differentiation), which was set to 1, and expressed as mean ± SEM. *, P < 0.05 vs. d 0; #, P < 0.05 vs. pCAGGS; , P < 0.05 vs. without transfection.

View larger version (19K): [in this window] [in a new window]   FIG. 5. The expression levels of adipocyte differentiation markers, C/EBP, C/EBPß, C/EBP, PPAR-, and aP2 mRNA during adipocyte differentiation in 3T3-L1 cells by simultaneous klotho protein administration. 1, Relative levels of C/EBP (A), C/EBPß (B) and C/EBP (C), PPAR- (D), and aP2 (E) mRNA expression in 3T3-L1 cells by simultaneous klotho protein administration (black circle) or control (white circle). 2, C/EBP (A), C/EBP (B), and -tubulin protein expression by simultaneous Klotho protein administration or control by Western blotting. Representative pictures are presented. Experiments were performed three times in independent cultures. Each experiment consisted of three wells for control and three wells for klotho protein incubation. Data are expressed as relative to the average value at d 0 (3T3-L1 cells before induction of differentiation), which was set to 1, and expressed as mean ± SEM. *, P < 0.05 vs. 0 d; #, P < 0.05 vs. control.

View larger version (20K): [in this window] [in a new window]   FIG. 6. Expression levels of adipocyte differentiation markers, C/EBP, C/EBPß, C/EBP, PPAR-, and aP2 mRNA during adipocyte differentiation in 3T3-L1 cells administered klotho protein 2 d after adipocyte induction. The relative levels of C/EBP (A), C/EBPß (B) and C/EBP (C), PPAR- (D), and aP2 (E) mRNA expression in 3T3-L1 cells by Klotho protein administration (black circle) or control (white circle). Klotho protein was administered 2 d after adipocyte induction. Experiments were performed three times in independent cultures. Each experiment consisted of three wells for control and three wells for klotho protein incubation. Data are expressed as relative to the average value at d 0 (3T3-L1 cells before induction of differentiation), which was set to 1, and expressed as mean ± SEM. *, P < 0.05 vs. control; , P < 0.05 vs. 3 d.

View larger version (88K): [in this window] [in a new window]   FIG. 7. The effect of klotho overexpression on adipocyte differentiation estimated by Oil Red O staining. A, Differentiated adipocytes were fixed and stained with Oil Red O. Representative pictures (magnification, x100) at d 8 are presented. Cells were photographed at x100 under light microscopy. B, Cells were transfected with pCAGGS (white circle) and klotho-pCAGGS (black circle) 3 d before adipocyte induction, and lipid accumulation was assessed by quantification of OD510 in destained Oil Red O with isopropanol. ANOVA showed significant different between pCAGGS and klotho-pCAGGS groups. P value in the figure indicates the results of paired Student’s t test in each day. Experiments were performed four times in independent cultures. Each experiment consisted of three wells for pCAGGS and three wells for klotho-pCAGGS transfection.*, P < 0.05 vs. 3T3-L1 cells transfected with pCAGGS.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we used 3T3-L1 cells (mouse fibroblast cells) for investigation of adipocyte differentiation. 3T3-L1 cells have been well established and frequently used for adipocyte differentiation study. As shown in Fig. 1B, klotho protein was detected in 3T3-L1 cells and up-regulated during adipocyte differentiation as well as mRNA expression. We also detected mRNA expression of klotho in mouse mesenteric fat tissue. Expression levels were similar to that of differentiated 3T3-L1 cells and TCMK-1 cells, kidney epithelial cells, normalized to 18S-rRNA (data not shown). Although klotho mRNA levels of 3T3-L1 cells were quite low compared with those of mouse kidney tissue (about 1–1000), our findings in protein expression in 3T3-L1 cells may indicate existence of pathophysiological role of klotho expression in fat cells. Considering the autocrine/paracrine system, the meaning of the expression of Klotho protein in 3T3-L1 cells is emphasized because our Western blot analysis using klotho antibody, KM2076, could not detect Klotho protein in the circulating blood.

The elevation of klotho mRNA expression started 24 h after induction by insulin, Dex, and IBMX, and decreased after its peak at d 3. This finding differs from those of previous work showing that klotho expression increased for 7 d during the period of differentiation (10). The first 4 d after induction are known to be a phase of transient proliferation; after this, time proliferation stops, and terminal differentiation and hypertrophy start. Thus, klotho expression may have an important role for transient proliferation after induction. Interestingly, the pattern of klotho mRNA expression in the process of differentiation is similar to that of C/EBPß, which induces C/EBP, PPAR-, and transient proliferation.

PPAR- is a strong promoter for adipocyte differentiation (23). Pioglitazone, a PPAR- agonist, increased klotho expression in 3T3-L1 cells that had been confluent for 48 h without induction medium. The increase of klotho mRNA expression began 12 h after incubation and its peak 24 h later. Despite the fact that the cells were not induced by insulin, Dex, and IBMX, klotho expression increased in the very early phase. Pioglitazone also accelerated expression of C/EBP and C/EBP, which is consistent with the previous report that pioglitazone accelerates adipocyte differentiation (24, 25). PPAR- is a strong promoter of insulin sensitivity, which increases klotho expression. Klotho has been shown to suppress the insulin/IGF-I pathway; however, the increase in klotho mRNA expression was temporary, and PPAR- did not increase klotho expression in subconfluent 3T3-L1 cells. These findings suggest that cell confluency is necessary for enhancement of klotho expression.

We tried to suppress the functional activity of klotho using siRNA. Transfection of siRNA is difficult when cells are confluent or preadipocyte, so we transfected it into subconfluent 3T3-L1 cells. The mRNA of adipocyte differentiation markers was suppressed only at 24 h after induction. This transient suppression may be because efficient suppression by siRNA persisted only for 5 d after transfection, 2 d after induction. These results are consistent with the idea that klotho functions in promotion of adipocyte differentiation.

Then, we investigated the role of klotho in adipocyte differentiation by using overexpression of the klotho gene. We found that klotho overexpression resulted in the increased expression of adipocyte differentiation markers in the early phase, indicating that klotho may promote adipocyte differentiation. A recent study found that klotho suppresses the insulin/IGF-I pathway (3), but there may be some disparity in results. The insulin/IGF-I pathway is known to be important in signaling to promote adipocyte differentiation (12). Suppression of insulin/IGF-I pathway does not seem like a complete explanation for the multiple aging-related phenotypes seen in klotho-disrupted mice; thus, klotho may function in a different pathway in adipocyte differentiation. We ensured that the endpoint was promotion of adipocyte differentiation by using Oil Red O fluorescence and by measuring GPDH activity.

Results using purified klotho recombinant protein indicated that klotho works as a hormonal factor in promoting adipocyte differentiation. Interestingly, mRNA elevation of adipocyte differentiation markers was not dependent on the timing of Klotho protein administration but on the time passed after adipocyte induction, suggesting that the role of klotho’s effects on adipocyte differentiation is important in the transient proliferation phase.

In conclusion, during the transient proliferation phase of adipocyte differentiation, klotho expression increased, and klotho promoted adipocyte differentiation, which may contribute partly to undetectable amount of white adipose tissue in klotho mutant mice. Klotho may play an important role in transient proliferation during adipocyte differentiation.

	Acknowledgments

 
We thank Ms. Seiko Kaji, Ms. Kazuko Iwasa, and Ms. Takako Tanigawa for excellent technical assistance and San Francisco Edit (http://www.sfedit.net) for assistance in editing this manuscript.

	Footnotes

 
This work was supported by the Osaka-Medical Research Foundation for Incurable Diseases (2000, 2005), by the NOVARTIS Foundation for Gerontological Research (2001), and by Grants-in-Aid for scientific research from the Ministry of Education, Science, Sports, Culture, and Technology of Japan (13670709, 2001–2002, and 15590342, 2003–2004).

Y.C., H.R., K.I., M.I, Y.M, J.O., I.K., and T.O. have nothing declare.

First Published Online May 18, 2006

Abbreviations: aP2, Fatty acid binding protein 4; C/EBP, CCAAT/enhancer-binding protein; Dex, dexamethasone; FBS, fetal bovine serum; GPDH, glycerol-3-phosphate dehydrogenase; IBMX, 1-methyl-3-isobutyl-xanthin; PPAR, peroxisome proliferator-activated receptor; siRNA, small interfering RNA.

Received December 2, 2005.

Accepted for publication May 5, 2006.

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