Metabolic Advantage of 25(OH)D3 versus 1,25(OH)2D3 Supplementation in Infantile Nephropathic Cystinosis-Associated Adipose Tissue Browning and Muscle Wasting

Manifestations of infantile nephropathic cystinosis (INC) often include cachexia and deficiency of circulating vitamin D metabolites. We examined the impact of 25(OH)D3 versus 1,25(OH)2D3 repletion in Ctns null mice, a mouse model of INC. Six weeks of intraperitoneal administration of 25(OH)D3 (75 μg/kg/day) or 1,25(OH)2D3 (60 ng/kg/day) resulted in Ctns−/− mice corrected low circulating 25(OH)D3 or 1,25(OH)2D3 concentrations. While 25(OH)D3 administration in Ctns−/− mice normalized several metabolic parameters characteristic of cachexia as well as muscle function in vivo, 1,25(OH)2D3 did not. Administration of 25(OH)D3 in Ctns−/− mice increased muscle fiber size and decreased fat infiltration of skeletal muscle, which was accompanied by a reduction of abnormal muscle signaling pathways. 1,25(OH)2D3 administration was not as effective. In conclusion, 25(OH)D3 supplementation exerts metabolic advantages over 1,25(OH)2D3 supplementation by amelioration of muscle atrophy and fat browning in Ctns−/− mice.


Introduction
Infantile nephropathic cystinosis (INC), a genetic chronic kidney disease (CKD), results from cystinosin (CTNS) mutations and involves the deposition of cystine crystals in multiple organs [1,2]. Children with INC present with myopathy and neuromuscular abnormalities such as swallowing difficulty. Currently, there are no known treatments to address these comorbidities [3,4]. We described the cachexic phenotype in Ctns null mice, an animal model of INC, with extensive fat browning and muscle atrophy [5]. White fat stores energy, whereas brown fat utilizes stored energy during thermogenesis to produce heat [6]. White fat browning (a process in which white adipocytes phenotypically change to brown-fat-like cells) has been implicated in the progression of cachexia, as demonstrated by recent studies [7][8][9][10]. The metabolism of skeletal muscle and brown fat are connected as brown fat modulates the function of skeletal muscle through the release of myostatin, a powerful inhibitor of muscle function [11]. Importantly, fat browning precedes muscle wasting in cancer and CKD [12,13]. Characterizing the complex interactions between various energy-wasting pathways involved in cachexia represents a key step towards establishing effective clinical therapies for this profound complication in patients with INC.

Repletion of 25-Hydroxyvitamin D3 Improves Energy Homeostasis in Ctns −/− Mice
In the second series of experiments, we utilized a food restrictive strategy to study the effects of restoring 25(OH)D3 versus 1,25(OH)2D3 levels in Ctns −/− mice without the

Repletion of 25-Hydroxyvitamin D 3 Improves Energy Homeostasis in Ctns −/− Mice
In the second series of experiments, we utilized a food restrictive strategy to study the effects of restoring 25(OH)D 3 versus 1,25(OH) 2 D 3 levels in Ctns −/− mice without the effects of different nutritional intake. Ctns −/− + Vehicle mice were fed ad libitum and we determined their daily ad libitum caloric intake. The other mouse groups received an energy intake amount equal to that of Ctns −/− + Vehicle ( Figure 1C). Serum chemistry of the mice is listed in Table 2. Replenishing serum 25(OH)D 3 concentration normalized weight gain, fat mass content, resting metabolic rate, lean mass content, and muscle function (shown by rotarod and grip strength) in Ctns −/− mice; whereas replenishing serum 1,25(OH) 2 D 3 concentration improved but not normalize these parameters in Ctns −/− mice ( Figure 1D Table 2. Serum and blood chemistry of mice. Twelve-month-old Ctns −/− mice and WT mice were treated with 25(OH)D 3 (75 µg/kg/day), 1,25(OH) 2 D 3 (60 ng/kg/day), or vehicle control (ethylene glycol) for six weeks. Ctns −/− + Vehicle mice were fed ad libitum whereas all other groups of mice were given the equivalent amount of energy intake as those of Ctns −/− + Vehicle mice. Results are expressed and analyzed as in Table 1 Figure 1. ns signifies not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Repletion of 25-Hydroxyvitamin D3 Increases Muscle Fiber Size in Ctns −/− Mice
While investigating the effect of vitamin D repletion on skeletal muscle morphology in Ctns −/− mice, we found that the average cross-sectional area of the gastrocnemius increased significantly when restoring the levels of 25(OH)D3 but not 1,25(OH)2D3 ( Figure  6). Average gastrocnemius cross-sectional area was measured (G). Results are expressed and analyzed as in Figure 1. ns signifies not significant, * p < 0.05, ** p < 0.01.   Average gastrocnemius cross-sectional area was measured (G). Results are expressed and analyzed as in Figure 1. ns signifies not significant, * p < 0.05, ** p < 0.01.

Molecular Mechanism of 25-Hydroxyvitamin D3 Repletion by RNAseq Analysis
In a previous study, we identified twenty different genes that play a role in energy metabolism, organismal injury and abnormalities, as well as the development and function of skeletal, muscular, and nervous systems by performing RNAseq analysis on gas-   Figure 3. ns signifies not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.
We showed the impact of 25(OH)D 3 repletion in correcting cachexia and in vivo muscle function in Ctns −/− mice. These results may have translational importance. Anorexia and increased energy use at rest are associated with poor survival in subjects on chronic dialysis [40,41].
UCPs regulates energy metabolism for the entire body [42]. Upregulation of adipose and muscle UCPs has been described in cachexia from different diseases and thought to be mechanistic involved in hypermetabolism in these disorders [43,44]. UCPs, mitochondrial inner membrane proteins, produce heat while ATPases, proton channels located in the same membrane, generate ATP. Increased expression of UCPS not only stimulates the process of thermogenesis but also inhibits the synthesis of ATP [42]. Compared to the repletion of 1,25(OH) 2 D 3 , 25(OH)D 3 repletion in Ctns −/− mice not only normalized fat UCP1 and muscle UCP3 levels but also significantly increased their ATP content. Murine fat and human cells all expressed VDR and 1α hydroxylase, the local enzyme that hydroxylates 25(OH)D 3 to 1,25(OH) 2 D 3 [45][46][47]. When mouse 3T3-L1 pre-adipocytes were incubated with 25(OH)D 3 , the media showed a buildup of 1,25(OH) 2 D 3 [48]. 25(OH)D 3 also binds to the UCP3 promoter region to modulate its expression in muscle [49]. WAT of Ctns −/− mice, there show upregulated thermogenic genes (Ppargc1α, Pgc1α, Cidea, Prdm16, and Dio2) (Figure 4), which was attenuated or normalized with 25(OH)D 3 repletion.
Additionally, we documented morphological features in skeletal muscle of mice by measuring fiber diameter and fat deposition in gastrocnemius muscle. In Ctns −/− mice, 25(OH)D 3 significantly improved muscle diameter and decreased fat deposition whereas 1,25(OH) 2 D 3 did not (Figures 6 and 7).
INC results from cystine accumulation primarily in kidney with many comorbidities [2,3]. Myopathy is prevalent in long term follow up studies in INC patients, including those who were treated with cysteamine. Gahl et al. [51] reported myopathy in 50% of 100 patients with INC; the incidence rising to 80% as the time of off-cysteamine therapy increased. Brodin-Sartoruius et al. reported myopathy in 22 out of 86 adult INC patients who were treated with cysteamine in a more recent long-term follow up study [52]. We measured muscle cystine content in our experimental animals. Muscle cystine content was significantly increased in Ctns −/− mice and repletion of 25(OH)D 3 or 1,25(OH) 2 D 3 did not change muscle cystine content in Ctns −/− mice (Figure 8). This would suggest that muscle wasting in INC is not the direct consequence of cystine accumulation.
Repletion of 25(OH) normalized or decreased muscle inflammatory cytokine expression in Ctns −/− mice ( Figure 5). Inflammation may interact with oxidative stress, abnormal autophagy, apoptosis, defective endocystic trafficking, impaired proteolysis as well as mitochondrial dysfunction in cystinotic cells [53,54]. We will plan future research to address these potential pathways.
Finally, we used RNAseq analysis to assess the muscle transcriptome. Importantly, 25(OH)D 3 , but not 1,25(OH) 2 D 3 , significantly improved the abnormal signature of muscle genes (13 upregulated and 5 downregulated) in Ctns −/− mice ( Figure 9). Ankrd2, Csrp3, Cyfip2, Fhl1, Ly6a, Spp1, and Tpm3 as well as Fos and Tbc1d1 are important determinants of muscle mass [19]. Mup1, Myl2, Pdk4, and Sln as well as Cidea and Sncg have been associated with energy metabolism. Supplementary Materials: The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Table S1: Immunoassay information for blood and serum chemistry, muscle adenosine triphosphate content as well as muscle and adipose tissue protein analysis.