Endocrinology. Apr 2012; 153(4): 1795–1805. Published online Jan 31, 2012. doi: 10.1210/en.2011-1878
PMCID: PMC3320267
Long-Term Fgf23 Deficiency Does Not Influence Aging, Glucose Homeostasis, or Fat Metabolism in Mice with a Nonfunctioning Vitamin D ReceptorCarmen Streicher, Ute Zeitz, Olena Andrukhova, Anne Rupprecht, Elena Pohl, Tobias E. Larsson, Wilhelm Windisch, Beate Lanske, and Reinhold G. Erben
Department of Biomedical Sciences (C.S., U.Z., O.A., A.R., E.P., R.G.E.), University of Veterinary Medicine, 1210 Vienna, Austria; Department of Medical Sciences (T.E.L.), Uppsala University, 751 85 Uppsala, Sweden; Department of Food Science and Technology (W.W.), University of Applied Life Sciences, 1180 Vienna, Austria; and Department of Developmental Biology (B.L.), Harvard School of Dental Medicine, Boston, Massachusetts 02215
Corresponding author.Address all correspondence and requests for reprints to: Reinhold G. Erben, M.D., D.V.M., Institute of Physiology, Pathophysiology, and Biophysics, Department of Biomedical Sciences, University of Veterinary Medicine, Veterinaerplatz 1, 1210 Vienna, Austria., E-mail:
It is still controversial whether the bone-derived hormone fibroblast growth factor-23 (FGF23) has additional physiological functions apart from its well-known suppressive actions on renal phosphate reabsorption and vitamin D hormone synthesis. Here we analyzed premature aging, mineral homeostasis, carbohydrate metabolism, and fat metabolism in 9-month-old male wild-type (WT) mice, vitamin D receptor mutant mice (VDR ) with a nonfunctioning vitamin D receptor, and Fgf23 /VDR compound mutant mice on both a standard rodent chow and a rescue diet enriched with calcium, phosphorus, and lactose. Organ atrophy, lung emphysema, and ectopic tissue or vascular calcifications were absent in compound mutants. In addition, body weight, glucose tolerance, insulin tolerance, insulin secretory capacity, pancreatic beta cell volume, and retroperitoneal and epididymal fat mass as well as serum cholesterol and triglycerides were indistinguishable between vitamin D receptor and compound mutants. In contrast to VDR and Fgf23 /VDR mice, which stayed lean, WT mice showed obesity-induced insulin resistance. To rule out alopecia and concomitantly elevated energy expenditure present in 9-month-old VDR and Fgf23 /VDR mice as a confounding factor for the lacking effect of Fgf23 deficiency on fat mass, we analyzed whole-body composition in WT, Fgf23 , VDR , and Fgf23 /VDR mice at the age of 4 wk, when the coat in VDR mice is still normal. Whole-body fat mass was reduced in Fgf23 mice but almost identical in WT, VDR , and Fgf23 /VDR mice. In conclusion, our data indicate that Fgf23 has no molecular vitamin D-independent role in aging, insulin signaling, or fat metabolism in mice.
The phosphaturic hormone fibroblast growth factor (FGF)-23 was discovered through genetic studies in patients suffering from a renal phosphate-wasting disease, autosomal dominant hypophosphatemic rickets (1). The mutations in the FGF23 gene found in autosomal dominant hypophosphatemic rickets patients interfere with cleavage and inactivation of FGF23, leading to excessive levels of intact circulating FGF23 (2, 3). Other phosphate-wasting disorders such as tumor-induced osteomalacia and X-linked hypophosphatemic rickets are also characterized by elevated serum levels of intact FGF23 (4, 5). FGF23 increases renal phosphate excretion by down-regulating proximal tubular membrane expression of the sodium phosphate cotransporters-2a and -2c (6–9). In addition, FGF23 suppresses renal proximal tubular 1α-hydroxylase expression, the key enzyme controlling synthesis of the biologically active vitamin D hormone 1α,25-dihydroxyvitamin D [1,25(OH) D ].
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FGF23 is secreted by osteocytes and osteoblasts in response to vitamin D and increased extracellular phosphate (10). Therefore, FGF23 is part of an endocrine feedback loop between bone and kidney, lowering circulating phosphate by a direct renal phosphaturic effect and an indirect mechanism down-regulating intestinal calcium and phosphorus absorption via suppression of 1,25(OH) D production.
FGF23 mediates its actions on renal tubular cells by signaling through the FGF receptor-1c and possibly other FGF receptors (11, 12). Binding of FGF23 to the ubiquitously expressed FGF receptors requires the transmembrane form of Klotho as an obligatory coreceptor (12), targeting the hormonal action of FGF23 to Klotho-expressing tissues. Klotho is mainly expressed in distal convoluted tubules in the kidney and in the choroid plexus in the brain (13). However, Klotho expression is also found in other tissues such as pituitary gland, parathyroid gland, inner ear, muscle, pancreas, gonads, and breast (reviewed in Ref. 14). It remains to be elucidated whether FGF23 signaling has a biological function in the latter tissues.
Ablation of Fgf23 function in gene-targeted (Fgf23 ) mice results in a complex phenotype characterized by severely impaired bone mineralization, hypercalcemia, hyperphosphatemia, highly elevated serum 1,25(OH) D levels due to increased renal 1α-hydroxylase expression, shortened life span, growth retardation, hypogonadism, emphysema, vascular calcifications, extensive soft tissue calcifications, atrophy of the intestinal villi, skin, thymus, and spleen together with hypoglycemia and profoundly increased peripheral insulin sensitivity (15–18).
Experiments in which the vitamin D signaling pathway was disrupted in Fgf23 mice suggested that the alterations in mineral and carbohydrate metabolism as well as the premature aging present in Fgf23 mice depend on intact signaling through the vitamin D receptor (VDR), and that the main physiological function of Fgf23 is its suppressive action on renal 1α-hydroxylase activity (17–19). However, an important limitation of these experiments is that they were all performed in fast-growing, 4- to 6-wk-old mice due to the short life span of Fgf23 mice. Data from older, nongrowing mice are lacking.
Human data suggest that FGF23 may have additional biological functions beyond mineral metabolism, which cannot be explained by current models of FGF23 action. For example, circulating FGF23 is positively associated with disease progression, heart hypertrophy, vascular calcifications, and mortality in human patients with chronic kidney disease (20). Even more intriguing, a recent study in two large cohorts of elderly subjects in Sweden reported a strong association between circulating FGF23 and obesity as well as adverse lipid metabolism (21). Other endocrine FGF such as FGF19 and FGF21 have antiobesity properties (22, 23), and FGF21 has been shown to inhibit growth hormone signaling (24). In addition, Klotho may have a role in adipocyte differentiation (25). Therefore, the question is whether the association between lipid metabolism and FGF23 reflects a causal link or just the fact that FGF23 is an excellent biomarker of phosphate and vitamin D metabolism. The link between obesity and low vitamin D status is well known (26), although the molecular mechanisms of this associations are poorly understood.
Here we sought to address the question whether Fgf23 is causally linked to aging as well as to carbohydrate and lipid metabolism by crossing Fgf23 mice (16) with VDR mutant mice (VDR ) mice characterized by a nonfunctioning VDR (27), and by analyzing glucose and lipid metabolism in 9-month-old wild-type (WT), VDR , and Fgf23 /VDR double-mutant mice. Due to the short life span of 4–8 wk in Fgf23mice, phenotypic analyses in older Fgf23 mice are not possible. We found that Fgf23 lacks a molecular role in aging and in the regulation of peripheral insulin sensitivity and lipid metabolism in 9-month-old VDR mice.
Materials and Methods
Animals
Heterozygous VDR mutant mice (27) and heterozygous Fgf23 mutant mice (16) on C57BL/6 genetic background (backcross generation F6) were mated to generate double-heterozygous animals. The double-heterozygous offspring from these matings were intercrossed to generate WT, Fgf23 , VDR , and Fgf23
/VDR mice. Genotyping of the mice was performed by multiplex PCR using genomic DNA extracted from tail as described (18). The mice were kept at 24 C with a 12-h light, 12-h dark cycle and were allowed free access to a normal rodent chow or the rescue diet and tap water. The rescue diet (Ssniff, Soest, Germany) containing 2.0% calcium, 1.25% phosphorus, 20% lactose, and 600 IU vitamin D per kilogram
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was fed starting from 16 d of age. This diet has been shown to normalize mineral homeostasis in VDR-ablated mice (27–29). At necropsy, the mice were exsanguinated from the abdominal vena cava under anesthesia with ketamine/xylazine (67/7 mg/kg, ip) for serum collection. The mice were not fasted before necropsy. The 4-wk-old mice for whole-body composition analysis were killed by cervical dislocation. All animal procedures were approved by the Ethical Committee of the University of Veterinary Medicine Vienna.
Biological chemistry
Serum triglycerides, cholesterol, aspartate aminotransferase activity, creatine kinase activity, total bilirubin, serum urea, creatinine, albumin, calcium, and phosphorus were analyzed on a Hitachi 766 autoanalyzer (Roche Diagnostics, Mannheim, Germany).
Glucose and insulin tolerance tests
Nine-month-old male WT, VDR , and Fgf23 /VDR mice on the normal and rescue diet were fasted individually on hunger grids overnight (12 h) with free access to tap water. On the next morning, glucose (1.5 mg/g body weight, dissolved in physiological saline at a concentration of 0.5 mmol/liter) was administered at time 0 by sc injection into the skinfold between the knee and the abdomen or by gavage. Blood glucose levels at 0, 10, 20, 30, 60, and 120 min were determined in whole-blood samples (2 μl, taken from a tail vein) using a standard test system (One-Touch Ultra; Lifescan, Neckargemünd, Germany). Each animal was used for both sc and oral glucose tolerance testing, with a 1-wk time interval between the tests. For insulin tolerance tests, the mice were deprived of food at time 0. Thereafter the mice were ip injected with insulin (0.75 IE/kg body weight; Humalog, Insulin Lispro; Eli Lilly and Co., Indianapolis, IN). Whole-blood glucose levels were measured at 0, 10, 20, 30, 60, and 120 min in tail vein samples (2 μl, One-Touch Ultra; Lifescan).
Organ histology
For organ histology, spleen, liver, intestine, thymus, lung, heart, aorta, kidney, pancreas, and skin were fixed in 4% paraformaldehyde overnight. Paraffin embedding, sectioning at 5 μm, and hematoxylin/eosin staining were carried out according to standard procedures. To detect ectopic calcifications, paraffin sections were stained with von Kossa and counterstained with nuclear fast red.
Islet histomorphometry
Organs were fixed in 4% paraformaldehyde overnight and were processed for paraffin histology and hematoxylin/eosin staining by routine methods. For immunohistochemical detection of insulin-expressing cells in paraffin sections of the pancreas, sections were deparaffinized, incubated for 15 min in 3% hydrogen peroxide in PBS to block endogenous peroxidase activity, and, after blocking with 20% rabbit serum, incubated for 2 h at room temperature with guinea pig antiporcine insulin antiserum diluted 1:2000 (Dako, Hamburg, Germany). Bound antibody was detected with peroxidase-conjugated rabbit anti-guinea pig IgG (Dako). Vector VIP (Vector Laboratories, Burlingame, CA) was used as enzyme substrate. Finally, the sections were counterstained with Mayer's hematoxylin. Histomorphometry of pancreatic islets was performed on antiinsulin-stained sections as described in detail previously (29).
Western blot analysis
Protein samples for Western blots were obtained from mouse brown and white adipose tissue as described (30). Tissue samples were lysed in lysis buffer (20 mM Tris; 0.25 mM sucrose; 5 mM EDTA; 1 mM EGTA, pH 7.5) with protease inhibitor cocktail (Sigma, Deisenhofen, Germany). After sonication (Bandolin Sonoplus GM70, Berlin, Germany), the lysate were centrifuged for 10 min at 2500 rpm. The supernatant was stored at −20 C. At least 50 μg proteins were loaded on 15% sodium dodecyl sulfate gels. Precision Plus protein dual-color standard (Bio-Rad Laboratories, Munich, Germany) was used as size marker. Blotting was accomplished with nitrocellulose membrane BA 85 (Schleicher & Schüell, Dassel, Germany) as described (30). The membranes were incubated with affinity-purified antimouse uncoupling protein-1 (UCP1) (1:1000) (Sigma) overnight at 4 C, followed by secondary horseradish peroxidase conjugated AB (1:5000, antirabbit IgG; GE Healthcare, Buckinghamshire, UK) for 1 h at room temperature. The immunoreaction was visualized using Pierce enhanced chemiluminescence Western blotting substrate on
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CL-Xposure films (GE Healthcare). For quantification, voltage-dependent anion channel (VDAC) and actin were consecutively detected on the same membrane. Anti-VDAC or anti-β-actin antibody were incubated for 1 h at room temperature. Recombinant UCP1 (31) was used in the Western blot analysis as a reference.
Statistical analyses
Statistics were computed using SPSS for Windows 17.0 (SPSS, Chicago, IL). The data were analyzed by one-way ANOVA followed by Student-Newman-Keuls multiple comparison test. Data from male and female mice were analyzed separately. Within one gender, we analyzed differences between the genotypes on the same diet as well as differences between groups with the same genotype on different diets. P < 0.05 was considered significant. The area under the curve for glucose tolerance tests was calculated using Prism 5.0 (GraphPad Software, La Jolla, CA). The data are presented as the mean ± SEM.
Results
Long-term lack of Fgf23 does not cause premature aging in mice with a nonfunctioning VDR
The premature aging-like phenotype in Fgf23 mice is characterized by growth retardation, hypogonadism, emphysema, vascular and soft tissue calcifications, and atrophy of the intestinal villi, skin, thymus, and spleen (17, 18). To determine whether long-term lack of Fgf23 gene function is associated with premature aging, we examined body weight, gross phenotype, organ weights, organ histopathology, and routine serum clinical chemistry in 9-month-old male WT, VDR and Fgf23 /VDR double-mutant mice. The mice were generated by interbreeding heterozygous Fgf23 mice with heterozygous gene-targeted mutant mice expressing a VDR with an intact hormone binding domain but lacking the first zinc finger necessary for DNA binding (VDR ) (18, 27). The life expectancy of Fgf23 mice is only 4–8 wk (15, 16). Therefore, all analyses in this study in 9-month-old mice had to be limited to WT, VDR , and Fgf23 /VDR compound-mutant mice.
Genetic ablation of vitamin D signaling in mice on a normal mouse diet results in severe secondary hyperparathyroidism (27, 32, 33). However, feeding of a so-called rescue diet rich in calcium, phosphorus, and lactose has been shown to normalize calcium homeostasis in mice with a nonfunctioning VDR (27, 28, 34). Therefore, we analyzed two cohorts of mice in this study: one on a normal diet and one on the rescue diet. An additional rationale for comparing two cohorts of mice on normal and rescue diet was that serum Fgf23 is low in VDR-ablated mice on normal diet but restored to WT control levels in VDR-ablated mice on rescue diet (35). The expected changes in calcium metabolism and circulating Fgf23 are shown in Table 1. The comparison between WT and VDR mice on rescue vs. normal diet permits assessing the direct effects of ablation of vitamin D signaling, vs. those mediated indirectly through secondary hyperparathyroidism. The comparison between VDR and Fgf23 /VDR compound mutant mice on normal diet is a comparison between a situation with low circulating Fgf23 in VDR mice and complete lack of Fgf23 in Fgf23 /VDR mice in the presence of hypocalcemia, whereas on rescue diet VDRmice with normal serum Fgf23 are compared with Fgf23 /VDR mice completely lacking Fgf23 in the setting of normocalcemia.
Relative to VDR-mutant mice, compound mutants had normal body weight and normal spleen weight on both the normal and the rescue diet (Fig. 1A). Male VDR and compound mutant mice on normal but not on rescue diet were hypocalcemic, whereas serum calcium levels did not differ between the genotypes in female mice on normal and rescue diet (Tables 2 and 3 and data not shown). As expected, due to the lacking phosphaturic factor Fgf23, compound mutants were hyperphosphatemic on either diet, relative to VDR mutants (Tables 2 and 3). Serum levels of creatinine, aspartate aminotransferase, creatine kinase, albumin, and urea were normal in VDR and compound mutants, suggesting normal liver and kidney function (Tables 2 and 3). In addition, compound mutants on either diet did not show organ atrophy, ectopic calcifications, or histopathological abnormalities in liver, kidney, spleen, heart, aorta, intestine, lung, and testicles (Fig. 1B and data not shown). These results indicate that the premature aging-like phenotype induced by Fgf23 deficiency is entirely caused by hypervitaminosis D and not by a putative molecular role of Fgf23 signaling in the aging process.
Peripheral insulin sensitivity is not influenced by Fgf23 deficiency
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Four-week-old Fgf23 mice are characterized by hypoglycemia and profoundly up-regulated peripheral insulin sensitivity (18). The pathophysiology behind this effect is unclear. It has been reported that the secreted form of the Klotho protein directly interacts with the IGF-I receptor and represses intracellular signals of insulin and IGF-I (36). It is conceivable that circulating Fgf23 may modulate the binding of secreted Klotho to the IGF-I receptor, thereby indirectly influencing insulin signaling. To explore the role of Fgf23 in the regulation of insulin sensitivity, we performed oral and sc glucose tolerance tests as well as insulin tolerance tests in 9-month-old male and female WT mice, VDR mutants, and Fgf23 /VDRdouble-mutant mice on normal and rescue diet. Only data from male mice are shown. It is evident from Fig. 2, A–C, that glucose and insulin tolerances were indistinguishable between VDR and compound mutants, indicating that lack of Fgf23 does not alter peripheral insulin sensitivity in VDR-ablated mice. VDR mutant mice had improved insulin sensitivity and glucose tolerance compared with WT mice (Fig. 2, A–C). This finding is at variance with our earlier report that 3-month-old VDR-mutant mice show impaired glucose tolerance and reduced insulin secretory capacity relative to WT controls (29). However, this conflict can easily be explained by the obesity seen in our 9-month-old WT mice (see below), resulting in insulin resistance, relative to VDR mice (Fig. 2C). In contrast, ablation of VDR function induces a lean phenotype and resistance to diet-induced obesity by a putative up-regulation of UCP1 in adipose tissue (37, 38). Compared with male mice, glucose and insulin tolerance tests yielded very similar results in female WT mice, VDR mutants, and Fgf23 /VDR double-mutant mice on normal and rescue diet (data not shown).
To characterize further glucose metabolism and to assess pancreatic β-cell mass, we measured insulin secretory capacity after an oral glucose challenge and performed a histomorphometric analysis of islets of Langerhans in male mice on rescue diet. As shown in Fig. 3A, there were no differences in insulin secretory capacity between the genotypes. WT mice had higher pancreas weight per body weight (BW) than VDR and compound mutants (Fig. 3B). Although there was a trend for higher mean islet area (Fig. 3C) and β-cell volume per kilogram BW (Fig. 3D) in WT vs. VDR and compound mutants, these changes did not reach statistical significance. Islet morphology and β-cell volume were identical in VDR and compound mutants, suggesting that the absence of Fgf23 had no influence on β-cell mass.
Fgf23 deficiency does not influence fat metabolism in VDR-ablated mice
To assess the effects of Fgf23 deficiency on fat metabolism, we analyzed fat mass and blood lipids in 9-month-old WT, VDR , and Fgf23 /VDR mice. Male and female VDR and Fgf23 /VDRmice had distinctly reduced retroperitoneal kidney fat mass, relative to WT mice (Fig. 4A). Epididymal fat mass in male VDR and Fgf23 /VDR mice was severalfold lower compared with WT mice (Fig. 4B). Male VDR and compound mutants on a normal diet showed lower serum cholesterol levels than WT controls (Fig. 4C). However, in male mice on rescue diet and in females on both diets, serum cholesterol was unchanged in VDR and compound mutants. Serum triglycerides did not differ between the genotypes on either diet (Fig. 4D). These data confirm previous studies reporting reduced fat mass and a lean phenotype in older VDR-ablated mice (37, 38). Furthermore, our study suggests that Fgf23 deficiency is not associated with alterations in fat mass or serum lipids in skeletally mature VDR-ablated mice.
It has been suggested earlier that the reason for the lean phenotype in older VDR-ablated mice is an up-regulation of UCP1 in white adipose tissue (37, 38). However, a possible confounding factor in these studies is that VDR-ablated mice develop alopecia by 2–4 months of age (27, 32, 33). To test the role of alopecia in the alterations in fat mass seen in aged VDR and compound mutants, we examined body composition in WT, Fgf23 , VDR , and Fgf23 /VDR mice at 4 wk of age, when the coat in VDR and compound mutants is still unchanged (Fig. 5A).
It is evident from Fig. 5B that 4-wk-old Fgf23 mice were characterized by dwarfism and distinct alterations in body composition, namely increased ash and protein content, and reduced fat content. However, body weight and body composition in VDR and Fgf23 /VDR mice were comparable with WT mice, showing that neither lack of a functioning VDR nor additional lack of Fgf23 influenced body composition in 4-wk-old mice. To examine whether the up-regulation in UCP1 expression observed in older VDR-ablated mice (37, 38) is secondary to the development of alopecia, we measured UCP1 concentrations in white and brown adipose tissue of 4-wk-old and 9-month-old WT and VDR mice by Western blotting. Figure 5, C and D, show that UCP1 concentrations in brown adipose tissue were similar
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in 4-wk-old and 9-month-old WT and VDR mutant mice. In white adipose tissue, we were unable to detect UCP1 in both genotypes in juvenile mice. In accordance with earlier reports (37, 38), we found increased UCP1 expression in white adipose tissue in 9-month-old VDR mice, relative to WT controls.
Discussion
It is still a matter of debate whether the bone-derived hormone FGF23 has additional physiological functions apart from its well-known suppressive actions on renal tubular phosphate reabsorption and renal 1α-hydroxlase expression (6–9, 18). For example, Fgf23-ablated mice are characterized by profoundly improved peripheral insulin sensitivity, a premature aging-like phenotype, and almost complete absence of sc fat (18). Moreover, recent human epidemiological studies indicated that circulating FGF23 is linked to obesity and lipid metabolism (21), and data from patients with chronic renal insufficiency showed that FGF23 serum levels are associated with disease progression, heart hypertrophy, and mortality (20). However, the current study clearly showed that Fgf23 lacks a vitamin D-independent molecular function in the aging process as well as in the regulation of carbohydrate and fat metabolism in mice.
One of our earlier studies in 4-wk-old Fgf23 /VDR double-mutant mice on rescue diet showed that ablation of the vitamin D signaling pathway completely rescued the premature aging-like phenotype in Fgf23 mice (18). A caveat of the latter study was that the mice were only 4 wk of age and that the rapid growth in these very young mice might mask any more subtle effects of Fgf23 on aging. However, the current experiment provides strong evidence that Fgf23 lacks a molecular role in the aging process in mice. Rather, the premature aging-like phenotype in Fgf23-ablated mice appears to be entirely due to hypervitaminosis D, leading to hypercalcemia and hyperphosphatemia. Although the exact pathophysiological mechanisms are poorly understood, the triad of chronic hypervitaminosis D, hypercalcemia, and hyperphosphatemia not only leads to vascular and organ calcifications but obviously also profoundly influences lipid and carbohydrate metabolism in juvenile Fgf23-ablated mice.
Epidemiological studies suggested that intact FGF23 serum levels are associated with body weight in humans (39) as well as with obesity and adverse lipid metabolism (21). In addition, circulating FGF23 is an independent predictor of disease progression, cardiovascular side effects, and mortality in patients with chronic kidney disease (20). However, our data indicate that this association is not caused by a direct effect of FGF23 on lipid or carbohydrate metabolism. Rather, our study suggests that these associations may reflect the fact that circulating FGF23 is an excellent biomarker of phosphate metabolism. High dietary phosphate or phosphate retention in chronic kidney disease stimulates osteocytic FGF23 secretion, which together with elevated extracellular phosphate suppresses renal 1α-hydroxylase. Therefore, elevated circulating FGF23 may be a sensitive biomarker of decreased renal vitamin D hormone production in health and disease. Hypovitaminosis D has been shown to be associated with obesity in young and elderly subjects (40, 41). However, the molecular mechanisms linking vitamin D and lipid metabolism are unclear at present.
Our study suggests that older VDR-ablated mice are not an apt model to study the role of vitamin D in lipid and carbohydrate metabolism because their lean phenotype makes it difficult to draw firm conclusions from experimental results. It is thought that VDR-ablated mice stay lean and are resistant to diet-induced obesity because of an up-regulation of UCP1 in white adipose tissue (37, 38), leading to increased nonshivering thermogenesis and higher energy expenditure. However, a possible major confounding factor in the interpretation of these studies is that VDR-ablated mice develop alopecia by 2–4 months of age (27, 32, 33). Alopecia increases energy loss through radiation and convection at temperatures below the neutral temperature, which is the case at the temperature of 22–24 C normally maintained in most mouse facilities, including ours. We also found a lean phenotype and increased UCP1 expression in white adipose tissue in 9-month-old VDR mice, whereas 9-month-old WT control mice showed obesity-induced insulin resistance, relative to lean VDR mice. In contrast, 4-wk-old VDR mice with intact coat were characterized by normal body composition, normal UCP1 expression in brown adipose tissue, and a lack of UCP1 expression in white adipose tissue. Therefore, our results challenge the notion that the VDR directly regulates UCP1 in adipocytes. Rather, the observed up-regulation of UCP1 in white adipose tissue of VDR-ablated mice at later ages (37, 38) is probably an adaptive mechanism driven by higher energy expenditure due to alopecia. Because 1α-hydroxylase-ablated mice do not develop alopecia (42), they might be a better model to investigate the function of the vitamin D hormone in carbohydrate and lipid metabolism.
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In conclusion, our data strongly suggest that the associations between circulating FGF23 and cardiovascular risk factors in patients with renal disease or with obesity and adverse lipid serum profile in normal subjects are not driven by a direct effect of FGF23 on lipid metabolism.
Acknowledgments
We thank C. Bergow and S. Hirmer for excellent technical assistance.
This work was supported by a grant from the University of Veterinary Medicine, Vienna, Austria (to R.G.E.). O.A. was supported by a postdoctoral fellowship from the University of Veterinary Medicine. B.L. was supported by National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases Grant DK073944.
Present address for T.E.L.: Department of Clinical Science, Intervention, and Technology, Karolinska Institutet, Stockholm, Sweden.
Present address for W.W.: Center of Life and Food Sciences Weihenstephan, Technische Universität München, Munich, Germany.
Disclosure Summary: The authors have nothing to disclose.
FootnotesAbbreviations:
BW Body weight FGF fibroblast growth factor 1,25(OH) D 1α,25-dihydroxyvitamin DVDR vitamin D receptor VDR VDR-mutant mice UCP1 uncoupling protein-1 VDAC voltage-dependent anion channel WT wild type.
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Figures and Tables
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Fgf23 deficiency does not cause premature aging in VDR-ablated mice. A, Body weight and spleen weight in 9-month-old male WT, VDR mutant (VDR ), and Fgf23 /VDR compound mutant mice on normal and rescue diet. Data represent mean ± SEM of seven to eight mice each. B, Von Kossa-stained paraffin sections of kidney, lung, and thoracic aorta in 9-month-old male WT, VDR , and Fgf23 /VDR compound mutant mice on rescue diet. Right panels in B show severe renal and aortic calcifications as well as lung emphysema in a 6-wk-old Fgf23 mouse on rescue diet for comparison. Original magnification, ×200. Sections were counterstained with nuclear fast red.
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All values are means ± SEM of eight to 11 animals in each group.
P < 0.05 vs. same genotype on normal diet by one-way ANOVA followed by Student-Newman-Keuls multiple comparison test.P < 0.05 vs. WT.P < 0.05 vs. VDR .
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Lack of effect of Fgf23 deficiency on glucose metabolism. A, Oral glucose tolerance tests in 9-month-old male WT, VDR , and Fgf23 /VDR mice on normal and rescue diet. Glucose (1.5 mg/g BW) was administered by gavage at time 0 after a 12-h fast. B, For sc glucose tolerance tests, the mice received 1.5 mg/g glucose sc at time 0 after a 12-h fast. C, Insulin tolerance tests in 9-month-old male WT, VDR , and Fgf23 /VDR mice on normal and rescue diet. Insulin (0.75 IE/kg) was injected ip at time 0. AUC, Area under the curve. Each data point in A, B, and C represents the mean ± SEM of six to eight mice each. *, P < 0.05 vs. WT, one-way ANOVA followed by Student-Newman-Keuls test.
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Insulin secretory capacity and histomorphometry of islets of Langerhans are unchanged in Fgf23/VDR compound mutants relative to VDR mutant mice. A, Serum insulin levels at baseline and 10 min after an oral glucose challenge (1.5 mg/g BW) in 9-month-old male WT, VDR , and Fgf23 /VDR mice on rescue diet. B–D, Relative pancreatic weight (B), mean islet size (C), and relative β-cell volume in male 9-month-old WT, VDR , and Fgf23 /VDR mice on rescue diet. Each data point in A–D represents the mean ± SEM of four to eight mice each. *, P < 0.05 vs. WT, one-way ANOVA followed by Student-Newman-Keuls test.
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Fgf23 deficiency does not influence fat metabolism in VDR-ablated mice. Fat of kidney capsule (A), epididymal fat (B), serum cholesterol (C), and serum triglycerides (D) in 9-month-old male and female WT, VDR , and Fgf23 /VDRmice on normal or rescue diet. The mice were not fasted before necropsy. Each data point represents the mean ± SEM of seven to eight mice each. *, P < 0.05 vs. WT, one-way ANOVA followed by Student-Newman-Keuls test.
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Fgf23 deficiency does not influence body fat content in juvenile VDR-ablated mice. A, Dwarfism in Fgf23 mice but lack of alopecia and normal body size in 4-wk-old VDR and Fgf23 /VDR mice on rescue diet. B, Body weight, ash in dry matter, protein in dry matter, and fat in dry matter in 4-wk-old male and female WT, VDR , Fgf23 , and Fgf23 /VDR mice on rescue diet. Each data point represents the mean ± SEM of five to nine mice each. *, P < 0.05 vs.WT, one-way ANOVA followed by Student-Newman-Keuls test. C and D, Representative Western blots of UCP1 protein expression in brown (BAT) and white adipose tissue (WAT) of 4-wk-old (C) and 9-month-old (D) WT and VDR mice (50 μg protein per lane). Anti-VDAC antibody was used as a control for mitochondrial number. Recombinant UCP1 was used as a reference.
Articles from Endocrinology are provided here courtesy of The Endocrine Society
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