Disordered metabolism in mice lacking irisin

Irisin is a product of fibronectin type III domain-containing protein (Fndc5) and is involved in the regulation of adipokine secretion and the differentiation of osteoblasts and osteoclasts. In this study, we aimed to determine whether irisin lacking affects glucose/lipid and bone metabolism. We knocked out the Fndc5 gene to generate irisin-lacking mice. Remarkable, irisin lacking was related to poor ‘browning response’, with a bigger size of the intraperitoneal white adipose cell and decreased a number of brown adipose cells in brown adipose of interscapular tissue. The irisin lacking mice had hyperlipidemia and insulin resistance, reduced HDL-cholesterol level, increased LDL-cholesterol level, and decreased insulin sensitivity. The lacking of irisin was associated with reduced bone strength and bone mass in mice. The increased number of osteoclasts and higher expression of RANKL indicated increased bone resorption in irisin lacking mice. The level of IL-6 and TNF-α also increased in irisin lacking mice. The results showed that irisin lacking was related to decreased ‘browning response’, glucose/lipid metabolic derangement, and reduced bone mass with increased bone resorption. Further studies are needed to confirm these initial observations and explore the mechanisms underlying the effects of irisin on glucose/lipid and bone metabolism.


Fndc5 knockout mice
Irisin is produced by proteolytic processing of a transmembrane receptor.Fndc5 is a 209-residue protein with an N-terminal 29-residue signal sequence followed by the irisin or putative fibronectin III domain, a linking peptide, a transmembrane domain, and a 39-residue cytoplasmic segment.Cleavage in the linking peptide region releases soluble irisin into the extracellular milieu.Therefore, we knocked out the Fndc5 gene to generate irisin lacking mice.Female Fndc5-heterozygous (+/−) mice were generated by View solid-biotech, Inc. (Beijing, China).Transcription activator-like effector nuclease (TALEN) technology was used to shear the DNA encoding the exon of the target gene.Fndc5 has six exons, and the coding gene of irisin is located in exon 3. Clipping the 18th and 19th nucleotides in exon three led to a frameshift mutation, which induced irisin deficiency in mice (Fig. 1A,B).Fndc5-heterozygous (+/−) female mice were bred with C57BL/6 WT male mice (Dossy, Chengdu, China) to produce heterozygous (+/−) male mice.Subsequently, female Fndc5-deficient (−/−) mice (Fig. 1C) were created by mating heterozygous (+/−) female mice with heterozygous (+/−) male mice.PCR-based genotyping analysis with tail genomic DNA was performed for Fndc5 using the following primers: 5′-CAT GTT TCC TTA GCT CTA CTGTG-3′ (forward) and 5′-GGA GAA AGC ATG CAT GGC AGTCT-3′ (reverse).It took nearly 2 years to generate 51 homozygous mice.Mice were housed with 4 to 5 animals per cage in a temperature-controlled environment on a 12-h light/dark cycle.All animals were given access to food (Dossy company, Chengdu, China) and water.After 24 weeks of old, the animals were sacrificed.The liver, spleen, kidney, intraperitoneal white adipose tissue (iWAT), and interscapular brown adipose tissue (iBAT) were weighted, and blood samples were collected the obit for serum isolation.Photomicrographs of hematoxylin and eosin-stained sections of iWAT and iBAT were analyzed with ImageJ software (NIH, MD) for measuring adipocytes size and area.The total adipocytes area was measured in the number of pixels and then converted to the metric system.The femur and tibia were dissected and divested of soft tissue for histology, microarchitecture analysis, and a three-point bending test.

Three-point bending test
The tibia was dissected and separated from adjacent tissues.All specimens were tested under a load applied at a constant rate of 20 mm/min to analyze the biomechanical strength at the mid-portion of the tibia using a threepoint bending test conducted with a bone strength testing device (Bose 3220 Series II, Massachusetts, USA) as described previously 14 .

Analysis of bone microarchitecture
We assessed trabecular microarchitecture in the distal femur by micro-computed tomography (μCT, Quantum GX, PerkinElmer, USA).The transverse μCT slices were evaluated in an area 1-3 mm to the distal growth plate.The X-ray source was set at a voltage of 90 kVp and a current of 88 μA, with a 0.5-mm aluminum filter.The scanning angular rotation was 180。, and the volume size was set at 10 μm.The trabecular bone region was identified by semi-manually contouring the trabecular bone in the ROI with the assistance of an auto-thresholding software algorithm (Analyze 12.0, PerkinElmer).Morphometric variables were computed from the binarized images using direct, 3D techniques we assessed the bone mineral density (BMD; g/cm 2 ), trabecular bone volume (bone volume to tissue volume ratio, BV/TV; %), trabecular number (Tb.N; mm −1 ), and connection density (Conn.D; 1/mm 3 ).

TRAP staining
The distal femur was fixed in 4% PFA fixation solution.After decalcifying in ethylene diamine tetra-acetic acid (EDTA) for seven days, the femur was longitudinally sectioned (5 μm thickness), and the sections were stained for TRAP.The cells expressing TRAP enzyme were stained pink 15 .The slides were scanned with a Nikon ECLIPSE Ti Slide Scanning System (Nikon, Japan), and image analysis was performed using ImageJ software (NIH, MD).

Immunohistochemistry
Femur sections were pretreated with 0.1% Tween in PBS for 30 min.Tissue sections were blocked in 5.0% BSA for 2 h before incubation with 5 μg/ml rabbit polyclonal antibody against OPG or RANKL overnight at 4 ℃.Then, the tissue sections were washed in TBST and incubated with HRP-conjugated secondary antibodies (Zen-Bioscience Company, China).The color was developed using diaminobenzidine (DAB) peroxidase substrate (Invitrogen, UK) for 2 min before counterstaining with hematoxylin (Solarbio, Beijing, China).Images were analyzed with the ImageJ software (NIH, MD) for quantifying the intensity of the reaction (Mean density = IOD Sum/Area Sum).

Serum levels of bone markers
The OCN, and TRAP levels in serum samples were measured using the ELISA kit (Elabscience, Wuhan, China).The ELISA assays were performed according to the manufacturer's manual, respectively.

Statistical analysis
All data are presented as the mean ± SEM.Differences in the mean values between two groups were assessed by two-tailed Student t-test.One-way and two-way ANOVA were used for data analysis of more than two groups, followed by Bonferroni's post hoc analysis.P < 0.05 was considered statistically significant.

Reduced browning response and hyperlipidemia in irisin lacing mice
In this study, we knocked out Fndc5 to generated irisin lacking mice (KO), and performed wild type mice (WT) as a control group.Although KO mice had significantly reduced body weight, they had an increased intraperitoneal white adipose tissue (iWAT) ratio, and a decreased interscapular brown adipose tissue (iBAT) ratio compared to WT mice (Fig. 2A; Table 1).
Besides, we stained adipose sections with H&E, and noted that KO mice had a bigger size of white adipocyte size (Fig. 2B,D).There was a weak 'browning response' in KO mice, displayed by fewer brown adipocytes in adipose tissue (Fig. 2C,E).To identify the effects of irisin on lipid metabolism, we measured the blood lipids of the mice.The results showed that KO mice had higher LDL-cholesterol levels (0.26 ± 0.02 mmol/L vs 0.15 ± 0.01 mmol/L, P < 0.001) and lower HDL-cholesterol levels (0.51 ± 0.04 mmol/L vs 0.68 ± 0.04 mmol/L, P < 0.001) compared to WT mice (Fig. 3C,D).The TG and TC concentrations were similar between the two groups (Fig. 3A,B).The liver, spleen, and kidney weights were comparable between the two groups (Table 1).

Reduced glucose tolerance and insulin sensitivity in irisin lacking mice
We performed a glucose tolerance test (GTT) and an insulin tolerance test (ITT) to evaluate glucose tolerance and insulin sensitivity.Our results showed that KO mice had poor glucose tolerance and significantly elevated glucose levels at 30 min following glucose challenge compared with those of WT mice (10.78 ± 0.25 mmol/L vs. 7.08 ± 0.22 mmol/L, P < 0.001) (Fig. 4A,B).We quantified the efficiency of insulin by its ability to reduce blood glucose levels in the ITT.The results showed that KO mice had decreased insulin sensitivity (3.80 ± 0.11 mmol/L vs. 2.79 ± 0.10 mmol/L, P < 0.001), showing insulin resistance (Fig. 4C).

Increased bone resorption in irisin lacking mice
In general, decreased bone mass is associated with decreased osteoblastic bone formation and, or increased osteoclastic bone resorption.OPG and RANKL are known to mediate osteoclast genesis.We investigated the dynamic bone resorption parameters in the distal femur.The results showed that KO mice had higher RANKL expression, with intense labeling (Fig. 6B), and the OPG signal were similar in KO and WT group (Fig. 6A).Also, we further investigated bone resorption parameters based on TRAP staining.Our results showed that KO mice had increased osteoclast numbers (Fig. 6C).In addition, we measured the bone formation and resorption parameters in serum, and the results indicated that KO mice had a higher TRAP level than WT mice (65.86 ± 4.70 U/L vs. 44.77± 2.48 U/L, P < 0.01) (Fig. 6D).The serum levels of OCN and ALP were comparable in KO and WT mice (Supplementary Figure 1).

Discussion
The adipokine irisin is involved in the dysregulation of adipokine secretion.It might lead to the development of excess adiposity and a decrease in insulin sensitivity and the regulation of the inflammatory response [16][17][18][19] .
In this study, we observed endocrine metabolism in irisin lacking mice.Our results demonstrated that irisin lacking mice showed poor 'browning response' , glucose/lipid metabolic derangements, and decreased bone strength and bone mass.WAT is the primary site of triglyceride storage, and BAT specializes in energy expenditure.Irisin is known to mediate the browning of WAT into BAT.BAT has drawn attention as a novel preventive and therapeutic target to treat obesity and metabolic diseases such as T2D.Our study showed an intraperitoneal fat accumulation, and a decreased 'browning response' in irisin lacking mice.Furthermore, our results revealed that the KO mice had glucose/lipid metabolic derangements, reduced glucose tolerance, and insulin resistance.These results were consistent with in vivo studies showing that irisin overexpression directly promotes Akt phosphorylation to improve glucose/lipid metabolism and insulin resistance in HFD mice 20 .In addition to the direct effects of irisin, there seem to be two potential reasons for insulin resistance in KO mice.One is that irisin lacking was associated with increased growth hormone (GH) concentration in KO mice (Supplementary Figure 2).GH is known to catabolize stored fat to release energy to promote growth in other tissues.However, recent studies indicated that the chronic stimulation of lipolysis by GH results in an increased free fatty acid (FFA) concentration in the systemic circulation.The excess FFA activates the TGF-β signaling pathway to induce insulin resistance through Smad-3-mediated downregulation of the Fndc5 gene 21,22 .Hence, a sustained release of high levels of GH significantly contributes to the development of insulin resistance by antagonizing the antilipolytic action of insulin.GH also mediates the deregulation of the FSP27-PPARγ axis, alters adipose tissue homeostasis and contributes to the development of insulin resistance 23 .Another reason is likely that, in our study, irisin deletion was related to the increased level of proinflammatory cytokine (IL-6 and TNF-α) in serum.In adipose tissue, IL-6 and TNF-α were the activators of the NF-κB pathway, linked to insulin resistance in obesity 24,25 .Studies have suggested that IL-6 production in abdominal adipose tissue is threefold higher than that of subcutaneous adipose tissue 25,26 .In the present study, the KO mice had abdominal fat accumulation, which indicated that IL-6 might be one of the factors that make abdominal adipose tissue a high-risk factor for the development of insulin resistance.
In bone metabolism, the results showed that KO mice had decreased bone strength and bone mass at 24 weeks of age, increased bone resorption.Our previous studies revealed that irisin promotes osteoblast proliferation and differentiation via the MAPK signaling pathway and inhibits differentiation in osteoclast precursor cells 11,12 .We also previously found that exogenously administered irisin has potential effects in resisting bone loss in OVX mice 13 .In the present study, irisin lacking was linked to increased osteoclast numbers and increased expression of RANKL, which indicated that irisin deficiency promoted bone resorption and resulted in sharp bone loss in KO mice.However, Kim et al. reported that Fndc5 null mice had higher trabecular bone mass than wild-type mice 27 .This discrepancy might be due to the animal model.They performed germline deletion of Fndc5(irisin residues 30-69 deletion, 70-112 remaining).In this study, we clipped the 18th, and 19th nucleotides in exon three led to a frameshift mutation, resulting in irisin residues 43-112 deletion.A recent study demonstrated that the flexible region of 55-58 and 106-108 residues, and C-terminal of irisin are vital for its activity 28 .Disrupting the dimerization of irisin might result in a partly reduced effect on cell differentiation.Therefore, our deletion of irisin (43-112 residues) disturbed salt bridges and C-terminal, severely affecting its physiological functions.Additionally, it is known that bone homeostasis of bone is tightly regulated by the balanced activities between bone-resorbing activity of osteoclast cells and bone-forming ability osteoblast cells 29 .This complicated process is governed by many endocrine factors.Rahman et al. presented the beneficial action of BAT and insulin-like growth factor 1(IGF-1) include positive effects on bone.Insulin-sensitive is also positively correlated with bone mass 30 .Thus, in this study, the poor 'brown response' , insulin resistance, and decreased serum level of IGF-1 (Supplementary Figure 2) were likely related to the reduced bone mass in mice lacking irisin.IGF-1 plays a critical role in developing the growing skeleton by establishing both longitudinal and transverse bone accrual 31 .A previous study indicated that older men with low serum IGF-1 have an increased fracture risk, and the association between serum IGF-1 and fracture risk is partly mediated by BMD 32 .In addition, irisin was an anti-inflammatory factor and reduced the secretion of proinflammatory cytokines (IL-6 and TNF-α) in 3T3 L1 cells [33][34][35][36] .In this study, the results showed that irisin lacking was related to the increased levels of IL-6 and TNF-α in KO mice.Firstly, excess IL-6 and TNF-α production increased the osteoclast-covered bone surface and decreased bone formation.Secondly, in the present study, the increased level of IL-6 and TNF-α were linked to a higher expression of RANKL in KO mice, which is consistent with the research showing that inflammation induces bone loss by increasing osteocyte protein expression of RANKL 37,38 .
To our knowledge, this was the first study to observe multiple metabolic processes in irisin lacking mice.We found poor 'browning response' , hyperlipidemia, insulin resistance, reduced bone mass, and increased inflammatory factors in irisin lacking mice.These results indicated metabolic disorders in irisin lacking mice, suggesting that irisin might be involved in multiple metabolic processes.However, our current results are initial observations, and are unable to elucidate the specific mechanism (e.g., signaling pathway) of irisin.Moreover, metabolism is a complex process; whether there are other factors that play a role together with irisin needs further studies.

Conclusion
Irisin lacking was related to poor 'brown response' , glucose/lipid derangement, and decreased bone mass in mice.The increased bone resorption induced bone loss in irisin lacking mice.This study opens the prospect of targeting irisin in improving metabolic derangement and preventing bone loss.

Table 2 .
Biomechanical strength of the tibia and three-point bending parameters in different groups.Data represent the mean ± SEM (n = 15).**P < 0.01 compared to WT.