MicroRNA‐188 regulates aging‐associated metabolic phenotype

Abstract With the increasing aging population, aging‐associated diseases are becoming epidemic worldwide, including aging‐associated metabolic dysfunction. However, the underlying mechanisms are poorly understood. In the present study, we aimed to investigate the role of microRNA miR‐188 in the aging‐associated metabolic phenotype. The results showed that the expression of miR‐188 increased gradually in brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT) of mice during aging. MiR‐188 knockout mice were resistant to the aging‐associated metabolic phenotype and had higher energy expenditure. Meanwhile, adipose tissue‐specific miR‐188 transgenic mice displayed the opposite phenotype. Mechanistically, we identified the thermogenic‐related gene Prdm16 (encoding PR domain containing 16) as the direct target of miR‐188. Notably, inhibition of miR‐188 expression in BAT and iWAT of aged mice by tail vein injection of antagomiR‐188 ameliorated aging‐associated metabolic dysfunction significantly. Taken together, our findings suggested that miR‐188 plays an important role in the regulation of the aging‐associated metabolic phenotype, and targeting miR‐188 could be an effective strategy to prevent aging‐associated metabolic dysfunction.

In our previous study, we showed that miR-188 plays crucial roles in regulating the aging-associated switch between osteoblast and adipocyte differentiation of bone marrow mesenchymal stem cells . However, a role for miR-188 in the regulation of the aging-associated metabolic phenotype remains to be investigated.
Therefore, in the present study, we aimed to investigate the role of miR-188 in the aging-associated metabolic phenotype. The results demonstrated that the expression of miR-188 increased gradually in mouse brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT) during aging. MiR-188 knockout mice were resistant to the aging-associated metabolic phenotype and had higher energy expenditure.
Adipose tissue-specific miR-188 transgenic (Tg) mice had the opposite phenotype. Notably, antagomiR-188-mediated inhibition of miR-188 expression in BAT and iWAT of aged mice ameliorated the aging-associated metabolic phenotype significantly. The results revealed that miR-188 regulates the aging-associated metabolic phenotype and could be a therapeutic target to treat aging-associated metabolic dysfunction.

| The expression of miR-188 increased gradually in mouse BAT and iWAT during aging
To determine the potential role of miR-188 in regulating the agingassociated metabolic phenotype, first we examined the expression of miR-188 in the BAT and iWAT of mice at different ages using quantitative real-time PCR (qPCR) analysis. The results showed that with increasing age, miR-188 expression increased gradually in the mouse BAT and iWAT (Figure 1a,b), which suggested that miR-188 plays a role in the regulation of the aging-associated metabolic phenotype.

| MiR-188 knockout mice were resistant to the aging-associated metabolic phenotype and had higher energy expenditure
To further investigate the potential role of miR-188 in regulating the aging-associated metabolic phenotype, we generated miR-188 null mice ( Figure S1a). There was no significant difference in body weight between the miR-188 null mice and their wild-type (WT) littermates during the first 10 months after birth ( Figure 2a). However, with increasing age, the body weights of the miR-188 null F I G U R E 1 The expression of miR-188 was gradually increased in BAT and iWAT of mice during aging. (a) The expression of miR-188 in BAT of WT mice at different ages as indicated. (b) The expression of miR-188 in iWAT of WT mice at different ages as indicated. Data are shown as means ± SEM (n = 6). Statistical significance was calculated by one-way ANOVA followed by Bonferroni posttest, *p < .05; **p < .01; ***p < .001   Energy homeostasis is maintained by the balance between food intake and energy expenditure (Kim, Seeley, & Sandoval, 2018;Xiao et al., 2017). Our findings showed that the aged miR- Thus, these findings suggested that the decreased body fat mass of the aged miR-188 null mice was largely caused by increased energy expenditure.

| Adipose tissue-specific miR-188 Tg mice were prone to develop the aging-associated metabolic phenotype and had lower energy expenditure
To further verify the findings reported above, we generated Tg mice that overexpressed miR-188 in an adipose tissue-specific manner ( Figure S2a). The body weights of the miR-188 Tg mice showed no significant differences compared with those of their WT littermates during the first 7 months after birth ( Figure 3a). However, with increasing age, the body weight of the miR-188 Tg mice gradually became higher than that of their WT littermates ( Taken together, these results suggested that the increased body fat mass of the aged adipose tissue-specific miR-188 transgenic mice was most likely caused by decreased energy expenditure.

F I G U R E 3
Adipose tissue-specific miR-188 Tg mice were prone to develop the aging-associated metabolic phenotype and had lower energy expenditure. Consistently, in the BAT of aged miR-188 knockout mice the protein levels of PRDM16 and UCP1 also increased, whereas in the aged miR-188 Tg mice, the protein levels of PRDM16 and UCP1 decreased (Figure 4g,h). These findings suggested that Prdm16 is a potential target of miR-188 in the regulation of the aging-associated metabolic phenotype.

| Administration of antagomiR-188 to aged mice ameliorated aging-associated metabolic phenotype and stimulated energy expenditure
The findings above suggested that miR-188 is a potential therapeutic target for aging-associated metabolic dysfunction. To inhibit the expression of miR-188 in the BAT and iWAT of aged mice, we injected antagomiR-188 to the tail veins of aged mice, as reported previously . Six months later, we found that antagomiR-188 injection significantly inhibited the expression of miR-188 in the BAT and iWAT of the aged mice ( Figure S3a). Monthly body weight measurements of the mice revealed that antagomiR-188 injection gradually decreased the body weight of the aged mice (Figure 5a). and Cidea were significantly increased in the BAT and iWAT of the antagomiR-188-injected mice (Figure 5p,q), and the protein levels of PRDM16 and UCP1 were also increased in the BAT of the antago-miR-188-injected mice compared with those in the controls ( Figure   S6h).
Taken together, these findings suggested that administration of antagomiR-188 ameliorated the aging-associated metabolic phenotype by increasing energy expenditure. And targeting miR-188 might be an effective way to prevent aging-associated metabolic dysfunction.

| D ISCUSS I ON
MiRNAs have been reported to participate in the regulation of a wide variety of metabolic diseases. However, a role for microRNAs in the regulation of the aging-associated metabolic phenotype has F I G U R E 4 Prdm16 is a potential target of miR-188 in the regulation of the aging-associated metabolic phenotype. (a) Schematic of the sequence that miR-188 targets the WT or mutated 3' UTR of Prdm16 mRNA. (b) The luciferase activity of Prdm16 WT or mutated 3' UTR reporter plasmids co-transfected with miR-188 mimics or miR-NC mimics in HEK293 cells as indicated. (c, d) The mRNA levels of Prdm16 in primary cultured brown adipocytes transfected with miR-188 mimics or miR-NC mimics. (e, f) The protein levels of PRDM16 in primary cultured brown adipocytes transfected with miR-188 mimics or miR-NC mimics. (g, h) The protein levels of PRDM16 and UCP1 in BAT of 18-month old miR-188 knockout mice and miR-188 Tg mice as indicated. Data are shown as means ± SEM (n = 5-7 in g, h), the cell experiments were repeated for at least three times. Statistical significance was calculated by Student's t test or two-way ANOVA, ***p < .001 not been reported. In our previous study, we demonstrated that miR-188 is an important regulator of aging-associated bone mass loss . Therefore, we questioned whether miR-188 also functions in the aging-associated metabolic phenotype.

AntagomiR-NC
The results of the present study showed that the expression of miR-188 gradually increased in the BAT and iWAT of mice during aging. For the WT mice, some metabolic changes gradually appeared during aging, including body weight gain and fat mass accumulation.
However, the aged mice with miR-188 knockout did not develop these phenotypes, while the adipose tissue-specific miR-188 Tg mice developed these phenotypes to a greater extent. These findings revealed that miR-188 exerts an important role in the regulation of the aging-associated metabolic phenotype.
Energy homeostasis is maintained by a balance between food intake and energy expenditure (Kim et al., 2018;Xiao et al., 2017).
In the aged miR-188 null mice and aged adipose tissue-specific miR-188 transgenic mice, the food intake was unchanged; however, the oxygen consumption and energy expenditure were significantly increased or decreased compared with those in the corresponding control mice, respectively. Under normal environmental conditions, energy expenditure occurs through physiological activities and thermogenesis (Cui et al., 2016;Deng et al., 2017;Wyler, Lord, Lee, Elmquist, & Liu, 2017). The mice's physiological activities were unchanged; however, the expression of thermogenesis-related genes increased or decreased significantly in the BAT and iWAT of aged miR-188 null mice or aged adipose tissue-specific miR-188 transgenic mice, respectively. Moreover, except for serum triglyceride, there was little difference between the genetic mice and their controls in terms of glucose and lipid metabolism ( Figure S3 and Figure S4).
Thus, these results suggested that miR-188 regulates the aging-associated metabolic phenotype largely by affecting the thermogenesis.
Further analysis identified Prdm16 as a downstream effector of miR-188, which may participate in the regulation of aging-associated metabolic phenotype. Prdm16 is a key transcription factor that regulates the expression of a panel of thermogenic program genes in brown adipocytes and beige adipocytes (Cohen et al., 2014;Harms et al., 2014) and plays an important role in maintaining iBAT and scWAT identity (Seale et al., 2011(Seale et al., , 2007. The results of the present study showed that miR-188 could bind directly to the 3' UTR of Prdm16 mRNA to inhibit its expression. This suggested that Prdm16 maybe the downstream target of miR-188 in the regulation of the aging-associated metabolic phenotype.
To further evaluate the therapeutic potential of targeting miR-188 in the treatment of aging-associated metabolic dysfunction, we injected antagomiR-188 into the aged mice via tail vein injection.
Administration of antagomiR-188 significantly inhibited the expression of miR-188 in the BAT and iWAT of the aged mice. Furthermore, antagomiR-188 injection ameliorated aging-associated metabolic phenotype significantly. Taken together, these findings revealed a role of miR-188 in the regulation of the aging-associated metabolic phenotype, suggesting that targeting miR-188 might be an effective way to prevent aging-associated metabolic dysfunction.

| Animals
C57BL/6J wild-type (WT) mice were obtained from Shanghai Laboratory Animals Co. Ltd (Shanghai, China). The miR-188 null mice were generated by transcription activator-like effector nuclease (TALEN) technique as reported previously . To generate adipose tissue-specific miR-188 transgenic (Tg) mice, first, the pre-miR-188 cDNA (synthesized by Shanghai sangon Co.) was subcloned into a plasmid containing Fabp4 (Ap2) promoter (Shi et al., 2014), resulting in Ap2-pre-miR-188 vector. Then, the Ap2-pre- (d-f) The aged male mice administrated with antagomiR-188 or antagomiR-NC for six months were sacrificed and the BAT, eWAT, and iWAT were isolated, (d) the gross morphology of BAT, eWAT, and iWAT; (e) the weight of BAT, eWAT, and iWAT; (f) hematoxylin and eosin staining of BAT, eWAT, and iWAT, scale bar: 100 μm. (g) The daily food intake of aged male mice administrated with antagomiR-188 or antagomiR-NC for six months. (h-n) The oxygen consumption, energy expenditure, respiratory exchange rate, and locomoter activity of aged male mice administrated with antagomiR-188 or antagomiR-NC for six months. (o) The rectal temperature of aged male mice administrated with antagomiR-188 or antagomiR-NC for six months. (p, q) The mRNA levels of a panel of thermogenic program genes in BAT and iWAT of aged male mice administrated with antagomiR-188 or antagomiR-NC for six months. All the data are shown as means ± SEM (n = 6-7). Statistical significance was calculated by Student's t test or two-way ANOVA, *p < .05; **p < .01 experiments were reviewed and approved by the Animal Care and

| Intravenous administration of miR-188 antagomir
The miRNA antagomir is a chemically modified, cholesterol conjugated, single-stranded RNA analog that complements the miR-NAs. It efficiently and specifically silences endogenous miRNAs.
AntagomiR-188 and its negative control (NC) were synthesized by RiboBio Co. For tail vein injection of antagomiR-NC or antago-miR-188, the aged mice were received antagomiR-188 once a week (10 mg/kg body weight, 0.2 ml for each injection) for six months before conducting metabolic parameters measurements. The antagomir negative control was administered at the same dose and injection intervals. The functional inhibition by the administered antagomirs in vivo was verified by qRT-PCR.

| Metabolic parameter measurements
The fat mass component and lean mass component of mice were measured by a nuclear magnetic resonance (NMR) system (Bruker, Rheinstetten, Germany). Indirect calorimetry was conducted in a comprehensive laboratory animal-monitoring system (Columbus Instruments, Columbus, OH), as described previously (Xiao et al., , 2016. Rectal temperature of mice was measured at 14:00 pm by a rectal probe attached to a digital thermometer (Physitemp, NJ, USA). The measurement of daily food intake was also conducted as reported previously (Xiao et al., , 2016.

| Histological analysis of tissues
Paraformaldehyde-fixed, paraffin-embedded sections of BAT, eWAT, and iWAT were stained with hematoxylin and eosin (H&E) for histology.

| Luciferase activity assays
The luciferase activity assays were conducted as reported previously (Li et al., 2009;Yang et al., 2017). Generally, the wild-type (WT) PRDM16 3'UTR firefly luciferase reporter plasmids or PRDM16 3'UTR firefly luciferase reporter plasmids with the potential miR-188 binding site mutated were co-transfected with miR-188 mimics or miR-NC mimics to the HEK293 cells, respectively. Renilla luciferase reporter plasmids were also transfected at the same time as internal control. 48 hr posttransfections, firefly and renilla luciferase activities were measured by a Dual-Glo Luciferase Assay System (Promega).

| Primary culture of brown adipocytes
The primary culture of brown adipocytes was performed as described previously (He, Tang, et al., 2018b;Hu et al., 2015). Briefly, the brown adipose tissue from three weeks old C57/BL6J mice was

| Western blot analysis
The Western Blot analysis was conducted as previously described (Li et al., , 2009, primary antibodies: anti-UCP1 was purchased from Cell Signalling Technology (#14670), anti-PRDM16 was purchased from Abcam (#ab202344), anti-β-actin was purchased from Proteintech (#HRP-60008). All validation information could be found on the manufacturer's website.

| RNA isolation and quantitative real-time PCR (qPCR)
The RNA isolation and qPCR analysis were performed as described previously (Li et al., , 2009). The primer pairs used in this study are listed in Table S1.

| Quantification and statistical analysis
All the results are expressed as means ± S.E.M. Each data point derived from qRT-PCR analysis represents an average of at least three technical replicates. The statistical significance of the differences between various treatments or groups was measured by either Student's t test or ANOVA followed by Bonferroni posttest. Data analyses were performed using GraphPad Prism 7.0. p < .05 was considered statistically significant, *p < .05; **p < .01; ***p < .001.

ACK N OWLED G EM ENTS
This work was supported by grants from National Natural Science

CO M PE TI N G FI N A N CI A L I NTER E S TS
The authors declare no competing financial interests.

AUTH O R S' CO NTR I B UTI O N
YZ.X and XH.L designed the experiments and wrote the manuscript; Y.H, Y.L and YZ.X performed most of the experiments; Y.X, Q.G, FL.Z, T.L and T.S helped to collect samples. YZ.X and XH.L is the guarantor of this work and, as such, has full access to all the data in this study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.