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A defect in Klotho gene expression in mice accelerates the degeneration of multiple age-sensitive traits. Here, we show that overexpression of Klotho in mice extends life span. Klotho protein functions as a circulating hormone that binds to a cell-surface receptor and represses intracellular signals of insulin and insulin-like growth factor 1 (IGF1), an evolutionarily conserved mechanism for extending life span. Alleviation of aging-like phenotypes in Klotho-deficient mice was observed by perturbing insulin and IGF1 signaling, suggesting that Klotho-mediated inhibition of insulin and IGF1 signaling contributes to its anti-aging properties. Klotho protein may function as an anti-aging hormone in mammals.
Klotho was originally identified as a mutated gene in a mouse strain that accelerates age-dependent loss of function in multiple age-sensitive traits (1). An insertional mutation that disrupts the 5′ promoter region of the Klotho gene resulted in a strong hypomorphic allele. Mice homozygous for the mutated allele (KL−/− mice) appeared normal until 3 to 4 weeks old but then began to manifest multiple age-related disorders observed in humans, including ectopic calcification, skin atrophy, muscle atrophy, osteoporosis, arteriosclerosis, and pulmonary emphysema. KL−/− mice suffered premature death around two months of age.
The Klotho gene encodes a single-pass transmembrane protein that is detectable in limited tissues, particularly the distal convoluted tubules in the kidney and the choroid plexus in the brain. Because a defect in the Klotho gene leads to systemic age-dependent degeneration, the Klotho protein may function through a circulating humoral factor that regulates the development of age-related disorders or natural aging processes (2). Notably, some single-nucleotide polymorphisms in the human KLOTHO gene are associated with altered life span (3) and altered risk for coronary artery disease (4), osteoporosis (5-7), and stroke (8).
Little is known about Klotho protein function and the molecular mechanism by which it suppresses the development of aging-like pheno-types. The extracellular domain of Klotho protein is composed of two internal repeats, KL1 and KL2, that share amino acid sequence homology to β-glucosidases of bacteria and plants (20 to 40% identity) (1). However, glucosidase activity is not present in recombinant Klotho protein (9), and the essential glutamate residue at the β-glucosidase active center is replaced with asparagine and alanine in KL1 and KL2, respectively (10). Here, we demonstrate that Klotho is an aging suppressor gene whose product functions as a hormone that inhibits intracellular insulin and IGF1 signaling.
We previously generated independent transgenic lines of mice that over-express Klotho under the control of the human elongation factor 1α promoter (1) (EFmKL46 and EFmKL48) (fig. S1). Here, we compared the life span of the transgenic mice with that of wild-type controls that are near-coisogenic by virtue of backcrossing onto the C3H background four times. Each line was previously confirmed to express functional Klotho protein from the transgene (1). Mice carrying the EFmKL46 or EFmKL48 transgenic alleles, fed ad libitum, outlived wild-type controls by 20.0 and 30.8%, respectively, in males (Fig. 1A) and by 18.8 and 19.0%, respectively, in females (Fig. 1B).
Caloric restriction is associated with increased longevity in various species (11). To assess whether mice overexpressing Klotho were restricting their own diets, we monitored food intake and oxygen consumption in transgenic and wild-type mice for 24 hours at 32 to 36 weeks of age. No significant differences in these parameters were observed (table S1). Small body size is also associated with extended longevity in diet-restricted mice and in mice that are mutant for pituitary or growth hormone receptor function (12, 13). However, we did not observe any substantial difference in growth between EFmKL46, EFmKL48, and wild-type mice (Fig. 1C). Both EFmKL46 and EFmKL48 breeding pairs generated fewer offspring than wild-type breeding pairs (Fig. 1D). As expected from the evolutionary theory of longevity, maximum fitness of the organism is a trade-off between life span and fertility (14). These data indicate that Klotho systemically modulates aging through mechanisms independent of food intake and growth, but potentially in association with reproduction.
Many genetic data demonstrate that inhibited insulin and IGF1 signaling extends life span in animals from C. elegans, to Drosophila, to mice (15-21). Because Klotho must mediate aging through effects of a systemic hormone, we investigated whether the Klotho gene is involved in the inhibition of insulin or IGF1 signaling. Mice defective in Klotho gene expression have reduced blood glucose and insulin levels coupled with enhanced sensitivity to insulin (22).
We compared glucose metabolism in the Klotho-overexpressing transgenic mice with wild-type animals. Blood glucose levels were normal in each transgenic line (Fig. 2A). However, male EFmKL46 and EFmKL48 mice had higher blood insulin levels than did wild-type males (Fig. 2B), suggesting that the male transgenic mice are somewhat insulin resistant. We directly assessed sensitivity to insulin with a hyperinsulinemic euglycemic clamp (23). As expected, male EFmKL46 and EFmKL48 mice required lower glucose infusion rates than did wild-type males to maintain normal blood glucose levels (Fig. 2C). Furthermore, insulin and IGF1 tolerance tests revealed significant attenuation in hypoglycemic response to injected insulin and IGF1 in male transgenic mice (Fig. 2, D and E). Although we were unable to detect insulin resistance in female transgenic mice (Fig. 2, C and D), they were significantly resistant to IGF1 (Fig. 2E). These studies demonstrate that Klotho overexpression induces resistance to insulin and IGF1.
The extracellular domain of Klotho is shed on the cell surface and detected in the blood and cerebrospinal fluid in mice and humans (24). Immunoblot analysis of plasma with the use of rabbit anti-Klotho antiserum demonstrated that the extracellular Klotho peptide can be detected in wild-type, EFmKL48, and EFmKL46 mice but not in KL−/− mice (fig. S2). Radioimmunoassay further demonstrated that Klotho peptide is ~100 pM in wild-type mice and about two times as high in the transgenic overexpression strains (fig. S3). The extra-cellular domain of Klotho may function as a hormone-like substance (2).
To assess the function of the Klotho extracellular peptide, we generated a soluble form of recombinant Klotho protein comprising the 952–amino acid extracellular domain, and determined whether this promoted insulin resistance when injected into mice. Intraperitoneal injection of insulin (0.5 U/kg) and purified Klotho extracellular peptide (10 μg/kg, Fig. 2F) in wild-type male and female mice attenuated the hypoglycemic response expected from insulin alone (Fig. 2G). Klotho peptide alone rapidly increased blood glucose levels in male wild-type mice and to a smaller extent in females (Fig. 2H). However, Klotho peptide injection did not induce significant changes in blood insulin and glucagon levels (fig. S4), suggesting that Klotho peptide inhibits insulin action directly in peripheral tissues.
To test whether Klotho antagonizes insulin at receptive cells, we determined whether recombinant Klotho peptide would reduce glucose uptake by blocking insulin binding to the insulin receptor. We measured cellular glucose uptake in cultured myoblastic cells (L6) incubated with or without insulin in the presence or absence of 100 pM of Klotho extracellular peptide. Klotho peptide suppressed insulin-induced glucose uptake by 55% without reducing the binding of [125I] insulin to the cells (fig. S5). Thus, Klotho does not appear to inhibit ligand-binding to the insulin receptor, suggesting that Klotho may block insulin action by disrupting one or more alternative insulin-dependent intracellular signaling pathways. Accordingly, we measured the potential for [125I]-labeled Klotho to bind directly to the cell surface. Hepatoma cells bound [125I] Klotho in a dose-dependent manner and saturated when the total Klotho concentration exceeded 600 pM, and unlabeled Klotho peptide inhibited the binding of [125I] Klotho (fig. S6). Together, these observations suggest that cells present a receptor at their surface other than the insulin receptor that binds to the Klotho peptide.
Because membrane-bound Klotho peptide must inhibit ligand activation of the insulin receptor within the cells, we investigated the influence of Klotho on insulin receptor signal transduction (25, 26). We incubated L6 cells or rat hepatoma cells (H4IIE) with recombinant Klotho peptide and insulin (10 nM) or IGF1 (10 nM). Klotho peptide did not inhibit the binding of [125I] insulin or [125I] IGF1 (fig. S5) but suppressed ligand-stimulated autophosphorylation of insulin and IGF1 receptors in a dose-dependent manner (Fig. 3, A and B). Additionally, Klotho reduced activation of signaling events downstream of receptor activation, including tyrosinephosphorylated insulin receptor substrate (IRS) 1 and 2, the association of the subunit of phosphoinositide 3–kinase p85 with IRS proteins (Fig. 3, A and B). Because the inhibitory effect of Klotho on insulin signaling was observed as early as 1 min after insulin stimulation (Fig. 3C), the decline in tyrosine-phosphorylated insulin and IGF1 receptors is unlikely due simply to the loss of receptors. Notably, Klotho peptide can inactivate active insulin receptors that were previously tyrosine phosphorylated by insulin stimulation. In H4IIE cells that were exposed to 10 nM insulin before adding Klotho peptide, Klotho suppressed tyrosine phosphorylation of the insulin receptor (Fig. 3D). We observed a similar effect on IGF1 receptor autophosphorylation in L6 cells with IGF1 before adding Klotho peptide (9). Importantly, the inhibitory effect of Klotho on autophosphorylation of receptor tyrosine kinases is specific. We observed no inhibitory effect of Klotho on the epidermal growth factor receptor and the platelet-derived growth factor receptor (fig. S7). Overall, Klotho appears to inhibit activation of the insulin and IGF1 receptor and to repress activated insulin and IGF1 receptors. Whether Klotho peptide functions by accelerating removal of tyrosine phosphorylation from the activated insulin receptor remains to be determined.
If the ability of Klotho to inhibit insulin and IGF1 signaling extends survival by retarding senescence, independent manipulations to inhibit insulin and IGF1 signaling may ameliorate some of the aging-like phenotypes in KL−/− mice. Accordingly, we crossed a loss-of-function mutation of IRS-1 into the KL−/− mice (27). Survival was improved in KL−/− mice heterozygous for an IRS-1 null allele (KL−/− IRS-1+/−) relative to KL−/− control mice (Fig. 4, A and B). In addition, KL−/− IRS-1+/− mice ameliorated many age-related pathologies typical of KL−/− mice, including arteriosclerosis, ectopic calcification, skin atrophy, pulmonary emphysema, and hypogonadism (Fig. 4, C to N). Heterozygosity of IRS-1 alone (KL+/+ IRS-1+/− littermates) appears to have no effect on survival and the age-progressive degeneration when compared with those factors in wild-type littermates during these experiments (9).
We previously reported that a defect in Klotho gene expression leads to a syndrome that may resemble premature aging (1). Here, we show that overexpression of Klotho can extend life span, and we suggest that Klotho functions as an aging suppressor gene in mammals. We found that the extra-cellular domain of Klotho protein circulates in the blood and binds to a putative cell-surface receptor. Klotho has marked effects on insulin physiology, apparently because it suppresses tyrosine phosphorylation of insulin and IGF1 receptors, which results in reduced activity of IRS proteins and their association with PI3-kinase, thereby inhibiting insulin and IGF1 signaling. Extended life span upon negative regulation of insulin and IGF1 signaling is an evolutionarily conserved mechanism to suppress aging (28). Klotho appears to be a peptide hormone to modulate such signaling and thereby mediate insulin metabolism and aging.