A high-fat diet stimulates fibroblast growth factor 23 formation in mice through TNFα upregulation

Background/objectives Bone-derived fibroblast growth factor 23 (FGF23) is a hormone that suppresses renal phosphate reabsorption and calcitriol (i.e., 1,25(OH)2D3) formation together with its co-receptor Klotho. FGF23- or Klotho-deficient mice suffer from rapid aging with multiple age-associated diseases, at least in part due to massive calcification. FGF23 is considered as a disease biomarker since elevated plasma levels are observed early in patients with acute and chronic disorders including renal, cardiovascular, inflammatory, and metabolic diseases. An energy-dense diet, which induces sequelae of the metabolic syndrome in humans and mice at least in part by enhancing pro-inflammatory TNFα formation, has recently been demonstrated to stimulate FGF23 production. Methods We investigated the relevance of TNFα for high-fat diet (HFD)-induced FGF23 formation in wild-type (tnf+/+) and TNFα-deficient (tnf−/−) mice. Results Within 3 weeks, HFD feeding resulted in a strong increase in the serum FGF23 level in tnf+/+ mice. Moreover, it caused low-grade inflammation as evident from a surge in hepatic Tnfα transcript levels. TNFα stimulated Fgf23 transcription in UMR106 osteoblast-like cells. Serum FGF23 was significantly lower in tnf−/− mice compared to tnf+/+ mice following HFD. Serum phosphate and calcitriol were not significantly affected by genotype or diet. Conclusions We show that HFD feeding is a powerful stimulator of murine FGF23 production through TNFα formation.


Introduction
The hormone fibroblast growth factor 23 (FGF23) is mainly produced by osteoblasts and osteocytes in the bone 1 . Its renal effects include inhibition of phosphate reabsorption and calcitriol formation 1,2 . Calcitriol is the biologically active form of vitamin D. The renal effects of FGF23 are mediated by a receptor which requires the protein α-Klotho (referred to as Klotho in the following) as an obligatory co-receptor 1 .
Klotho was originally discovered in 1997 as an antiaging protein [3][4][5] . Klotho-deficient mice have an extremely short life span of a few weeks only and exhibit many disorders associated with aging in humans 3 . FGF23deficient mice have a similar phenotype 6 . Both mouse strains suffer from drastically elevated plasma levels of phosphate and calcitriol due to the primary renal effect of FGF23 and Klotho. Importantly, the premature aging of Klotho-or FGF23-deficient mice is also a direct or indirect consequence of the hyperphosphatemia of the mice since maintaining them on a low phosphate or low vitamin D diet normalizes their life span 7

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A high plasma FGF23 level has been found in patients with various acute and chronic disorders including renal (acute kidney injury, chronic kidney disease), cardiovascular (coronary heart disease, myocardial infarction, atrial fibrillation), inflammatory, and metabolic diseases 8 . The role of FGF23 in chronic kidney disease is established best: Plasma FGF23 is elevated before a marked decrease of glomerular filtration rate (GFR), and it exhibits a strong positive correlation with mortality, hypertrophy of the left ventricle, and disease progression 9 . Therefore, it is presently being considered as a valuable disease biomarker. However, it is yet incompletely understood whether and to which extent FGF23 contributes to pathophysiological processes rather than merely indicating them. At least, FGF23 has been shown to induce hypertrophy of the left ventricle independently of Klotho 10 .
Metabolic syndrome is characterized by hypertension, glucose intolerance, dyslipidemia, as well as obesity, and affects millions of patients world-wide and represents a significant health burden particularly in industrialized countries 16 . Although the complex pathophysiological processes have not yet been uncovered completely, it is clear that an imbalance between caloric needs and intake is the predominant factor. In mice, a diet rich in fats (high-fat diet (HFD)) induces metabolic syndrome [17][18][19] . Low-grade inflammation associated with metabolic syndrome is relevant especially for the development of glucose intolerance 20 . In this respect, pro-inflammatory cytokines derived from adipose tissue or the liver are a major source of inflammation in metabolic syndrome. Among those cytokines, TNFα has been found to play a predominant role 21 . Interestingly, an energy-dense diet has recently been demonstrated to upregulate the production of FGF23 in rats 22 .
Here, we sought to define the role of metabolic syndrome-associated TNFα production in HFD-induced FGF23 formation.

Animals and treatments
All animal experiments were conducted according to the German law for the welfare of animals and were approved by the authorities of the state of Saxony-Anhalt. Experiments were performed in TNFα-deficient (tnf −/− ) mice (from The Jackson Laboratory, Sulzfeld, Germany; Stock No: 005540; the generation and genotyping is available on the website of The Jackson laboratory) and in age-and sex-matched wild-type mice (tnf +/+ ) fed a control diet (Ssniff, Soest, Germany; standard diet for maintenance V1534).
At the age of 8-10 weeks, the mice were fed a HFD containing 70% kcal from fat (Altromin, Lage, Germany; C1090-70) for 3 weeks, and the body weight was recorded weekly. The animals had free access to food and tap water. Serum was taken before and on the last day of the treatment. The exact number of mice and the number of replications is provided in the figure legends. For all animal experiments, no randomization was used, no blinding was done, and no statistical test was applied to estimate the sample size.

Serum parameters
To obtain blood specimens, the animals were lightly anesthetized with ether, and blood was drawn into heparinized capillaries by puncturing the retro-orbital plexus. Since the entire procedure takes less than a minute, it is unlikely to have a significant impact on our study. Serum concentrations of intact FGF23 and calcitriol were determined by ELISA kits (Immutopics, San Clemente, CA, USA; IDS, Frankfurt am Main, Germany). Inorganic phosphate was measured by a photometric method (Biocon ® Diagnostik, Vöhl/Marienhagen, Germany).

Cell culture
Cell culture was performed as previously described 23 . Briefly, UMR106 rat osteosarcoma cells (ATCC, Manassas, VA, USA) were cultured in DMEM high glucose medium (Gibco, Grand Island, NY, USA) supplemented with 10% FCS (Gibco) and 100 U/ml penicillin/100 µg/ml streptomycin (Gibco) under standard culture conditions. After 24 h, the cells were treated with or without TNFα (Sigma-Aldrich) for different periods.

Statistics
Data are provided as means ± SEM, n represents the number of independent experiments or number of mice per group, respectively. All data were tested for significance using the tests indicated in the figure legends. For serum FGF23, normal distribution was assumed. The data meet the assumptions of the respective tests. Variance was similar between the groups apart from the data in Fig. 2B and Fig. 4E. Therefore, Welch's correction was applied in these cases. Only results with p < 0.05 were considered statistically significant.

Results
At the age of 8-10 weeks, we started to feed wild-type mice (tnf +/+ ) a HFD ad libitum for 3 weeks. Similar to what has recently been demonstrated in rats 22 , the HFD caused a strong increase (by almost four times) in the serum intact FGF23 level (Fig. 1).
HFD feeding and subsequent adipose tissue accumulation are associated with subclinical inflammation and the generation of the key pro-inflammatory cytokine TNFα. Hence, we found that feeding HFD indeed resulted in a significant increase in liver Tnfα mRNA expression levels ( Fig. 2A) in tnf +/+ mice pointing to HFD-associated lowgrade inflammation. Moreover, also adipose tissue Tnfα mRNA expression levels (Fig. 2B) tended to be higher in tnf +/+ mice on HFD, a difference, almost reaching statistical significance (p = 0.106).
Next, we carried out cell culture experiments with UMR106 osteoblast-like cells to test whether TNFα is capable of stimulating FGF23 production as has been shown for IDG-SW3 cells 15 . According to Fig. 3a, a 24 h incubation with TNFα resulted in a dose-dependent upregulation of Fgf23 mRNA transcript levels in UMR106 cells with significance at 5 and 10 ng/ml TNFα. The time dependence for the effect of 5 ng/ml TNFα is illustrated in Fig. 3B.
Our last series of experiments explored whether the HFD-induced FGF23 production is dependent on TNFα formation. To this end, we compared tnf +/+ mice with tnf −/− mice. On control diet, the serum intact FGF23 concentration was not significantly different between tnf +/+ mice and tnf −/− mice (Fig. 4A). However, after 3 weeks of feeding the HFD, the serum intact FGF23 level was significantly different between the genotypes being nearly 50% lower in tnf −/− mice compared to tnf +/+ mice (Fig. 4A). Serum calcitriol was not significantly different between tnf −/− and tnf +/+ mice on either control or HFD, but was significantly lower in a group of HFD-fed mice compared to mice on control diet (Fig. 4B). Similarly, the serum phosphate concentration was not significantly affected by neither genotype nor diet (Fig. 4C). On control diet, no significant difference between the body weight of tnf +/+ mice and tnf −/− mice could be observed (Fig. 4D). Fig. 1 High-fat-diet-induced increase in the serum FGF23 concentration in tnf +/+ mice. Arithmetic means ± SEM of the serum intact FGF23 concentration (n = 3 mice, no replication) in tnf +/+ mice before (control) and after 3 weeks of feeding a high-fat diet (HFD); **p < 0.01 (paired, two-tailed t-test) However, the HFD resulted in significantly stronger weight gain in tnf +/+ mice than in tnf −/− mice (Fig. 4D, E).

Discussion
According to our study, the stimulatory effect of a HFD on FGF23 formation was significantly blunted in genetargeted mice devoid of pro-inflammatory TNFα (tnf −/− ). This result suggests that a HFD stimulates FGF23 production in large part by inducing low-grade inflammation.
It is well established that energy-dense diets including a HFD favor the development of metabolic syndrome characterized by insulin resistance, dyslipidemia, obesity, and hypertension 24,25 . This pathophysiological condition is associated with systemic low-grade inflammation 26 . In particular, a pivotal role for the pro-inflammatory cytokine TNFα in the development of obesity-induced insulin resistance has been demonstrated 21 .
Inflammation has emerged as a powerful factor driving FGF23 production 11 . Our study demonstrates that TNFα upregulated Fgf23 transcript levels in UMR106 osteosarcoma-like cells. Importantly, TNFα is effective through transcription factor NF-κB 15 and in line with this, NF-κB has also been demonstrated to enhance FGF23 synthesis 14 .
Elevated serum FGF23 concentrations are observed in acute and chronic renal, metabolic, and cardiovascular diseases 8 . Most of these clinical conditions are associated with inflammation. Therefore, similar to HFD feeding, these disorders may at least in part be effective in stimulating FGF23 production by enhancing the production of pro-inflammatory cytokines. Fig. 3 TNFα upregulated Fgf23 transcripts in UMR106 cells. Arithmetic means ± SEM of relative Fgf23 mRNA abundance (relative to Tbp mRNA) in UMR106 cells incubated for 24 h without (white bar) or with (black bars) TNFα at the indicated concentration (A; n = 5) or incubated with TNFα (5 ng/ml) for the indicated periods (B; n = 6); *p < 0.05 and **p < 0.01 (one-way ANOVA followed by Dunnett's multiple comparisons test) Fig. 2 High-fat-diet-induced increase in liver Tnfα transcript levels in tnf +/+ mice. Arithmetic means ± SEM (n = 7 mice per group, one replication) of relative hepatic (A; unpaired, two-tailed t-test) and adipose tissue (AT). (B; unpaired, two-tailed t-test with Welch's correction) Tnfα mRNA abundance (relative to Gapdh or 18S mRNA) in tnf +/+ mice on control diet and on high-fat diet (HFD) for 3 weeks. *p < 0.05 On control diet, the serum concentration of intact FGF23 was not significantly different between tnf +/+ mice and tnf −/− mice although a tendency toward lower FGF23 in tnf −/− mice was apparent. A pro-inflammatory milieu in HFD-treated animals, however, resulted in strong TNFα-dependent FGF23 generation.
Since an increase in serum FGF23 has been observed very early in some chronic disorders including chronic kidney disease, FGF23 has been suggested as a biomarker 27 . According to our results, an increase in serum FGF23 by almost four times was observed after 3 weeks of HFD feeding, a relatively short period as evident from a moderate increase in total body weight by only some 10% in tnf +/+ mice. Therefore, lower FGF23 may also indicate a better metabolic profile of an individual.
A major effect of FGF23 is the inhibition of renal calcitriol formation thereby lowering the serum calcitriol concentration 1 . Elevated FGF23 formation in HFD-fed mice could therefore be expected to decrease the serum calcitriol concentration. We did not, however, observe a significant difference in the serum calcitriol between the genotypes, although a tendency toward higher values in tnf −/− mice was obvious.
Taken together, our study demonstrates that a HFD stimulates FGF23 production at least in part by inducing TNFα formation.