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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-gamma 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)alpha, C/EBPbeta, C/EBPdelta, peroxisome proliferator-activated receptor-gamma, 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/EBPalpha and C/EBPdelta 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.

Homozygous Klotho mutant (kl-/-) mice exhibit a variety of phenotypes resembling human aging, including arteriosclerosis, infertility, skin atrophy, osteoporosis, and short life span. Calcium abnormality, one of the phenotypes in kl-/- mice, is thought to be due to the elevated gene expression of 25-hydroxyvitamin D3 1alpha-hydroxylase in the kidney. We studied 25-hydroxy-vitamin D3 1alpha-hydroxylase gene expression using a Klotho plasmid that we had previously constructed for Klotho protein production. It was found that Klotho protein medium upregulated cAMP and the PKC pathway, and suppressed 25-hydroxyvitamin D3 1alpha-hydrox-ylase in kidney cells. However, both cAMP and PKC are known to elevate 25-hydroxyvitamin D3 1alpha-hydroxylase gene expression, therefore, another unknown calcium regulation pathway using Klotho protein medium might exist. Furthermore, we found that activation of the PKC pathway by Klotho was observed only in the kidney and testis, where the Klotho gene is expressed, although activation of the cAMP pathway was observed in any kind of cell. These data suggest that calcium regulation through 25-hydroxyvitamin D3 1alpha-hydroxylase by Klotho depends on non-cAMP and a non-PKC pathway and that the Klotho protein may have different signaling pathways, depending on the Klotho gene expression in different cells and organs.

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Objective: Mice deficient in the klotho gene exhibit a syndrome resembling premature human aging. A recent report also suggested that klotho transgenic mice exhibited a long lifespan, which shows that klotho is an antisenescence gene. Previously, klotho has been reported to improve endothelial dysfunction, and also to have a preventive effect against oxidative stress. In the present study, we investigated the effect of klotho gene delivery on blood pressure and oxidative stress in vivo. Methods:Klotho plasmid was injected into the tail vein of mice and spontaneous hypertensive rats over 5 s. Results:Klotho gene delivery upregulated manganese superoxide dismutase protein expression and total superoxide dismutase activity in the aorta of mice compared with the control. It upregulated nitric oxide production, and downregulated lipid peroxide concentration in the serum of mice. When klotho plasmid was administered to spontaneously hypertensive rats, superoxide dismutase activity in the kidney and liver was significantly increased, and lipid peroxide concentration in the kidney and liver was significantly decreased, compared with the control. Klotho gene delivery in spontaneously hypertensive rats did not alter systolic blood pressure. Conclusion:These results suggest that klotho gene infusion into the tail vein of mice and rats has a suppressive effect against oxidative stress. These findings may provide a new insight into the therapeutic potential of klotho gene delivery in vivo to regulate oxidative stress.