Ute Zeitz

Impaired insulin secretory capacity in mice lacking a functional vitamin D receptor

It was the aim of this study to further explore the functional role of vitamin D in the endocrine pancreas. By gene targeting, we have recently generated mice in which a lacZ reporter gene is driven by the endogenous vitamin D receptor (VDR) promoter. These mice express a functionally inactive mutant VDR. Pancreatic islets but not exocrine pancreas cells showed strong lacZ reporter gene expression in mutant mice. To rule out possible influences of hypocalcemia on pancreatic endocrine function, a rescue diet enriched with calcium, phosphorus, and lactose was fed to wild‐type (WT) and VDR mutant mice. The rescue diet normalized body weight and mineral homeostasis in VDR mutants. In glucose tolerance tests, baseline blood glucose levels were unchanged in fasting VDR mutants. However, blood glucose was elevated after oral or subcutaneous glucose loading, and maximum serum insulin levels were reduced by ~60% in VDR mutants vs. WT mice on either diet. In addition, insulin mRNA levels were decreased in VDR mutant mice on both diets, whereas pancreatic β cell mass, islet architecture, and islet neogenesis were normal. These findings clearly establish a molecular role of the VDR in pancreatic insulin synthesis and secretion in vivo.

FGF23 acts directly on renal proximal tubules to induce phosphaturia through activation of the ERK1/2–SGK1 signaling pathway

Fibroblast growth factor-23 (FGF23) is a bone-derived endocrine regulator of phosphate homeostasis which inhibits renal tubular phosphate reabsorption. Binding of circulating FGF23 to FGF receptors in the cell membrane requires the concurrent presence of the co-receptor αKlotho. It is still controversial whether αKlotho is expressed in the kidney proximal tubule, the principal site of phosphate reabsorption. Hence, it has remained an enigma as to how FGF23 downregulates renal phosphate reabsorption. Here, we show that renal proximal tubular cells do express the co-receptor αKlotho together with cognate FGF receptors, and that FGF23 directly downregulates membrane expression of the sodium-phosphate cotransporter NaPi-2a by serine phosphorylation of the scaffolding protein Na+/H+ exchange regulatory cofactor (NHERF)-1 through ERK1/2 and serum/glucocorticoid-regulated kinase-1 signaling.

Inhibition of Receptor Activator of NF-κB Ligand by Denosumab Attenuates Vascular Calcium Deposition in Mice

Osteoporosis and vascular calcification frequently coincide. A potential mediator of bone metabolism and vascular homeostasis is the triad cytokine system, which consists of receptor activator of nuclear factor-kappaB (RANK) ligand (RANKL), its receptor RANK, and the decoy receptor osteoprotegerin. Unopposed RANKL activity in osteoprotegerin-deficient mice resulted in osteoporosis and vascular calcification. We therefore analyzed the effects of RANKL inhibition by denosumab, a human monoclonal antibody against RANKL, on vascular calcium deposition following glucocorticoid exposure. Prednisolone pellets were implanted into human RANKL knock-in (huRANKL-KI) mice, which unlike wild-type mice are responsive to denosumab. No histomorphological abnormalities or differences in aortic wall thickness were detected between wild-type and huRANKL-KI mice, regardless of treatment with prednisolone, denosumab, or both. However, concurrent treatment with denosumab reduced aortic calcium deposition of prednisolone-treated huRANKL-KI mice by up to 50%, based on calcium measurement. Of note, aortic calcium deposition in huRANKL-KI mice was correlated negatively with bone mineral density at the lumbar spine (P = 0.04) and positively with urinary excretion of deoxypyridinoline, a marker of bone resorption (P = 0.01). In summary, RANKL inhibition by denosumab reduced vascular calcium deposition in glucocorticoid-induced osteoporosis in mice, which is further evidence for the link between the bone and vascular systems. Therefore, the prevention of bone loss by denosumab might also be associated with reduced vascular calcification in certain conditions.

Prevention of glucocorticoid‐induced bone loss in mice by inhibition of RANKL

OBJECTIVE RANKL has been implicated in the pathogenesis of glucocorticoid-induced osteoporosis. This study was undertaken to evaluate the efficacy of denosumab, a neutralizing monoclonal antibody against human RANKL (hRANKL), in a murine model of glucocorticoid-induced osteoporosis. METHODS Eight-month-old male homozygous hRANKL-knockin mice expressing a chimeric RANKL protein with a humanized exon 5 received 2.1 mg/kg of prednisolone or placebo daily over 4 weeks via subcutaneous slow-release pellets and were additionally treated with phosphate buffered saline or denosumab (10 mg/kg subcutaneously twice weekly). Two groups of wild-type mice were also treated with either prednisolone or vehicle. RESULTS The 4-week prednisolone treatment induced loss of vertebral and femoral volumetric bone mineral density in the hRANKL-knockin mice. Glucocorticoid-induced bone loss was associated with suppressed vertebral bone formation and increased bone resorption, as evidenced by increases in the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, TRAP-5b protein in bone extracts, serum levels of TRAP-5b, and urinary excretion of deoxypyridinoline. Denosumab prevented prednisolone-induced bone loss by a pronounced antiresorptive effect. Biomechanical compression tests of lumbar vertebrae revealed a detrimental effect of prednisolone on bone strength that was prevented by denosumab. CONCLUSION Our findings indicate that RANKL inhibition by denosumab prevents glucocorticoid-induced loss of bone mass and strength in hRANKL-knockin mice.

FGF23 promotes renal calcium reabsorption through the TRPV5 channel

αKlotho is thought to activate the epithelial calcium channel Transient Receptor Potential Vanilloid‐5 (TRPV5) in distal renal tubules through its putative glucuronidase/sialidase activity, thereby preventing renal calcium loss. However, αKlotho also functions as the obligatory co‐receptor for fibroblast growth factor‐23 (FGF23), a bone‐derived phosphaturic hormone. Here, we show that renal calcium reabsorption and renal membrane abundance of TRPV5 are reduced in Fgf23 knockout mice, similar to what is seen in αKlotho knockout mice. We further demonstrate that αKlotho neither co‐localizes with TRPV5 nor is regulated by FGF23. Rather, apical membrane abundance of TRPV5 in renal distal tubules and thus renal calcium reabsorption are regulated by FGF23, which binds the FGF receptor‐αKlotho complex and activates a signaling cascade involving ERK1/2, SGK1, and WNK4. Our data thereby identify FGF23, not αKlotho, as a calcium‐conserving hormone in the kidney.

Vitamin D Is a Regulator of Endothelial Nitric Oxide Synthase and Arterial Stiffness in Mice

The vitamin D hormone 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] is essential for the preservation of serum calcium and phosphate levels but may also be important for the regulation of cardiovascular function. Epidemiological data in humans have shown that vitamin D insufficiency is associated with hypertension, left ventricular hypertrophy, increased arterial stiffness, and endothelial dysfunction in normal subjects and in patients with chronic kidney disease and type 2 diabetes. However, the pathophysiological mechanisms underlying these associations remain largely unexplained. In this study, we aimed to decipher the mechanisms by which 1,25(OH)2D3 may regulate systemic vascular tone and cardiac function, using mice carrying a mutant, functionally inactive vitamin D receptor (VDR). To normalize calcium homeostasis in VDR mutant mice, we fed the mice lifelong with the so-called rescue diet enriched with calcium, phosphate, and lactose. Here, we report that VDR mutant mice are characterized by lower bioavailability of the vasodilator nitric oxide (NO) due to reduced expression of the key NO synthesizing enzyme, endothelial NO synthase, leading to endothelial dysfunction, increased arterial stiffness, increased aortic impedance, structural remodeling of the aorta, and impaired systolic and diastolic heart function at later ages, independent of changes in the renin-angiotensin system. We further demonstrate that 1,25(OH)2D3 is a direct transcriptional regulator of endothelial NO synthase. Our data demonstrate the importance of intact VDR signaling in the preservation of vascular function and may provide a mechanistic explanation for epidemiological data in humans showing that vitamin D insufficiency is associated with hypertension and endothelial dysfunction.

FGF23 Regulates Bone Mineralization in a 1,25(OH)2 D3 and Klotho-Independent Manner.

Fibroblast growth factor-23 (Fgf23) is a bone-derived hormone, suppressing phosphate reabsorption and vitamin D hormone (1,25(OH)2 D3 ) production in the kidney. It has long been an enigma why lack of Fgf23 or of Klotho, the coreceptor for Fgf23, leads to severe impairment in bone mineralization despite the presence of hypercalcemia and hyperphosphatemia. Using Fgf23(-/-) or Klotho(-/-) mice together with compound mutant mice lacking both Fgf23 or Klotho and a functioning vitamin D receptor, we show that in Klotho(-/-) mice the mineralization defect is solely driven by 1,25(OH)2 D3 -induced upregulation of the mineralization-inhibiting molecules osteopontin and pyrophosphate in bone. In Fgf23(-/-) mice, the mineralization defect has two components, a 1,25(OH)2 D3 -driven component similar to Klotho(-/-) mice and a component driven by lack of Fgf23, causing additional accumulation of osteopontin. We found that FGF23 regulates osteopontin secretion indirectly by suppressing alkaline phosphatase transcription and phosphate production in osteoblastic cells, acting through FGF receptor-3 in a Klotho-independent manner. Hence, FGF23 secreted from osteocytes may form an autocrine/paracrine feedback loop for the local fine-tuning of bone mineralization.

Long-Term Fgf23 Deficiency Does Not Influence Aging, Glucose Homeostasis, or Fat Metabolism in Mice with a Nonfunctioning Vitamin D Receptor

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(Δ/Δ)) with a nonfunctioning vitamin D receptor, and Fgf23⁻/⁻/VDR(Δ/Δ) 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(Δ/Δ) and Fgf23⁻/⁻/VDR(Δ/Δ) 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(Δ/Δ) and Fgf23⁻/⁻/VDR(Δ/Δ) mice as a confounding factor for the lacking effect of Fgf23 deficiency on fat mass, we analyzed whole-body composition in WT, Fgf23⁻/⁻, VDR(Δ/Δ), and Fgf23⁻/⁻/VDR(Δ/Δ) mice at the age of 4 wk, when the coat in VDR(Δ/Δ) mice is still normal. Whole-body fat mass was reduced in Fgf23⁻/⁻ mice but almost identical in WT, VDR(Δ/Δ), and Fgf23⁻/⁻/VDR(Δ/Δ) mice. In conclusion, our data indicate that Fgf23 has no molecular vitamin D-independent role in aging, insulin signaling, or fat metabolism in mice.

Klotho Lacks a Vitamin D Independent Physiological Role in Glucose Homeostasis, Bone Turnover, and Steady-State PTH Secretion In Vivo

Apart from its function as co-receptor for fibroblast growth factor-23 (FGF23), Klotho is thought to regulate insulin signaling, intracellular oxidative stress, and parathyroid hormone (PTH) secretion in an FGF23 independent fashion. Here, we crossed Klotho deficient (Kl−/−) mice with vitamin D receptor (VDR) mutant mice to examine further vitamin D independent functions of Klotho. All mice were fed a rescue diet enriched with calcium, phosphorus, and lactose to prevent hyperparathyroidism in VDR mutants, and were killed at 4 weeks of age after double fluorochrome labeling. Kl−/− mice displayed hypercalcemia, hyperphosphatemia, dwarfism, organ atrophy, azotemia, pulmonary emphysema, and osteomalacia. In addition, glucose and insulin tolerance tests revealed hypoglycemia and profoundly increased peripheral insulin sensitivity in Kl−/− mice. Compound mutants were normocalcemic and normophosphatemic, did not show premature aging or organ atrophy, and were phenocopies of VDR mutant mice in terms of body weight, bone mineral density, bone metabolism, serum calcium, serum phosphate, serum PTH, gene expression in parathyroid glands, as well as urinary calcium and phosphate excretion. Furthermore, ablation of vitamin D signaling in double mutants completely normalized glucose and insulin tolerance, indicating that Klotho has no vitamin D independent effects on insulin signaling. Histomorphometry of pancreas islets showed similar beta cell volume per body weight in all groups of animals. In conclusion, our findings cast doubt on a physiologically relevant vitamin D and Fgf23 independent function of Klotho in the regulation of glucose metabolism, bone turnover, and steady-state PTH secretion in vivo.

Fgf23 and parathyroid hormone signaling interact in kidney and bone.

Fibroblast growth factor-23 (FGF23) is a bone-derived hormone, suppressing renal phosphate reabsorption and vitamin D hormone synthesis in proximal tubules, and stimulating calcium reabsorption in distal tubules of the kidney. Here, we analyzed the long term sequelae of deficient Fgf23 signaling on bone and mineral metabolism in 9-month-old mice lacking both Fgf23 or Klotho and a functioning vitamin D receptor (VDR). To prevent hypocalcemia in VDR deficient mice, all mice were kept on a rescue diet enriched with calcium, phosphate, and lactose. VDR mutants were normocalcemic and normophosphatemic, and had normal tibial bone mineral density. Relative to VDR mutants, Fgf23/VDR and Klotho/VDR compound mutants were characterized by hypocalcemia, hyperphosphatemia, and very high serum parathyroid hormone (PTH). Despite ∼10-fold higher serum PTH levels in compound mutants, urinary excretion of phosphate and calcium as well as osteoclast numbers in bone remained unchanged relative to VDR mutants. The increase in plasma cAMP after hPTH(1-34) injection was similar in all genotypes. However, a 5-day infusion of hPTH(1-34) via osmotic minipumps resulted in reduced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) in bone and kidney of Fgf23/VDR and Klotho/VDR compound mutants, relative to VDR and WT controls. Similarly, the PTH-mediated ERK1/2 phosphorylation was reduced in primary osteoblasts isolated from Fgf23 and Klotho deficient mice, but was restored by concomitant treatment with recombinant FGF23. Collectively, our data indicate that the phosphaturic, calcium-conserving, and bone resorption-stimulating actions of PTH are blunted by Fgf23 or Klotho deficiency. Hence, FGF23 may be an important modulator of PTH signaling in bone and kidney.