Abstract

Hyperuricaemia (HUA) significantly increases the risk of metabolic syndrome and is strongly associated with the increased prevalence of high serum free fatty acids (FFAs) and insulin resistance. However, the underlying mechanisms are not well established, especially the effect of uric acid (UA) on adipose tissue, a vital organ in regulating whole-body energy and FFA homeostasis. In the present study, we noticed that adipocytes from the white adipose tissue of patients with HUA were hypertrophied and had decreased UCP1 expression. To test the effects of UA on adipose tissue, we built both in vitro and in vivo HUA models and elucidated that a high level of UA could induce hypertrophy of adipocytes, inhibit their hyperplasia and reduce their beige-like characteristics. According to mRNA-sequencing analysis, UA significantly decreased the expression of leptin in adipocytes, which was closely related to fatty acid metabolism and the AMPK signalling pathway, as indicated by KEGG pathway analysis. Moreover, lowering UA using benzbromarone (a uricosuric agent) or metformin-induced activation of AMPK expression significantly attenuated UA-induced FFA metabolism impairment and adipose beiging suppression, which subsequently alleviated serum FFA elevation and insulin resistance in HUA mice. Taken together, these observations confirm that UA is involved in the aetiology of metabolic abnormalities in adipose tissue by regulating leptin-AMPK pathway, and metformin could lessen HUA-induced serum FFA elevation and insulin resistance by improving adipose tissue function via AMPK activation. Therefore, metformin could represent a novel treatment strategy for HUA-related metabolic disorders.

Introduction

Uric acid (UA) is a final oxidation product of purine catabolism. The serum UA balance is maintained by dietary uptake, production and excretion of purines [1]. Hyperuricaemia (HUA) is associated with various diseases, such as coronary artery disease, hypertension, diabetes mellitus, cerebrovascular disease, chronic kidney disease and gout [2–4]. In addition, clinical trials have shown that a high serum UA level also significantly increases the risk of metabolic syndrome [5,6], such as hypertriglyceridemia and high free fatty acids (FFAs) [7], and results in insulin resistance [8]. However, the effects and exact mechanisms remain unclear.

White adipose tissue (WAT) is a vital organ in maintaining lipid homeostasis through a fine-tuned system of uptake, esterification (lipogenesis) and release of FFAs (lipolysis), the so-called ‘triacylglycerol cycling’ [9,10]. When energy is in surplus, lipid is stored through the enlargement of adipocytes (hypertrophy) and increase in adipocyte numbers (hyperplasia) [11], thereby keeping FFAs and blood glucose below toxic levels [12]. Moreover, WAT could be shifted to beige or so-called brite (brown-like-in-white) adipose tissue, which possesses the brown-like feature of energy dissipation through heat production with the use of FFAs as fuel through activating uncoupling protein 1 (UCP1), the brown adipose tissue-specific protein [13]. In such a way, adipose tissue ensures that the serum level of FFAs, which serve as a key energy source for skeletal muscle, myocardium and other major organs, is within the normal range. However, when adipose tissue is dysfunctional, serum FFAs increase, and associated metabolic complications such as insulin resistance occur [14].

Adipocytes express urate-anion exchanger 1 (URAT1), a transporting protein on cell responsible for the UA reabsorption [15], which indicated that UA could be absorbed by adipocytes and influence the function of adipose tissue. Clinical studies have found that obese individuals have a higher UA level than healthy controls [16], and elevated serum UA is closely associated with the accumulation of visceral fats [17]. William et al. demonstrated that UA could increase the expression of monocyte chemotactic protein-1 (MCP-1) and inhibit the production of adiponectin in cultured adipocytes, which could be a potential mechanism of the insulin resistance in patients suffering from metabolic syndrome [18]. However, the exact effect of UA on affecting the metabolic function of adipose tissue, as well as the underlying molecular mechanisms, is poorly understood.

Metformin is a widely used oral anti-diabetic agent that can exert a beneficial effect on improving lipid metabolism [19], repress de novo lipogenesis in hepatocytes and prevent hepatic steatosis by AMP-activated protein kinase (AMPK) activation [20]. AMPK is a key regulator in different cellular metabolic pathways. Thus, we hypothesized that metformin could lessen HUA-induced serum FFA elevation and insulin resistance by improving adipose tissue function via AMPK activation. To verify this hypothesis, we built both in vitro and in vivo HUA models to investigate the effects as well as major mechanism of UA on the metabolic function of adipocyte tissue, and to test if AMPK activation by metformin is a novel potential treatment strategy for HUA-related metabolic disorders.

Materials and methods

Clinical samples

Ethical Committee of the First Affiliated Hospital of Harbin Medical University approved the present study, and all the patients received and signed a written informed consent (No. 202040). Adipose tissue samples were obtained from 16 patients who scheduled for laparotomy surgery due to external abdominal hernia or abdominal trauma at the First Affiliated Hospital of Harbin Medical University, including 8 patients with normal levels of serum UA (males: 149-416 μmol/l; females: 89-357 μmol/l), and 8 patients with HUA diagnosis (males > 420 μmol/l, females > 360 μmol/l). In our study, patients with diabetes, inflammatory disease, infection and cancer were excluded. Human adipose tissue samples were obtained from nearly the same location in patients undergoing laparotomy. Histological specimens were prepared and used for H&E and UCP1 immunohistochemistry. Details on patients’ characteristics are depicted in Table 1.

Table 1
Characteristics of Non-HUA and HUA subjects
VariableNon-HUA (n=8)HUA (n=8)P value
Age (years) 47.7 ± 4.9 41.9 ± 5.5 0.45 
Males % 5 (63%) 6 (75%) 0.59 
BMI (kg/m224.1 ± 0.4 26.2 ± 1.0 0.06 
Serum UA (μmol/l) 285.9 ± 19.9 537.9 ± 51.2 0.0004 
Serum TG (mmol/l) 1.3 ± 0.2 2.6 ± 0.5 0.04 
Serum FFA (mmol/l) 0.6 ± 0.05 1.0 ± 0.12 0.02 
VariableNon-HUA (n=8)HUA (n=8)P value
Age (years) 47.7 ± 4.9 41.9 ± 5.5 0.45 
Males % 5 (63%) 6 (75%) 0.59 
BMI (kg/m224.1 ± 0.4 26.2 ± 1.0 0.06 
Serum UA (μmol/l) 285.9 ± 19.9 537.9 ± 51.2 0.0004 
Serum TG (mmol/l) 1.3 ± 0.2 2.6 ± 0.5 0.04 
Serum FFA (mmol/l) 0.6 ± 0.05 1.0 ± 0.12 0.02 

Animals and diets

The animal work was completed in the Animal Laboratory of the First Affiliated Hospital of Harbin Medical University and was approved by the Animal Care and Use Committee of the First Affiliated Hospital.

Six-week-old male C57BL/6J mice weighing 20 ± 2 g (Vital River Laboratories, Beijing, China) were housed under a 12:12-h light–dark cycle at constant 22°C. The mice were randomly divided into two groups: the control group (n=10) was fed a chow diet (10% of kcal from fat) and drank normal water; the HUA group (n=30) was fed a high-fat diet containing excess fat (46%), high fructose (17.5%) and sucrose (17.5%) and drank 10% fructose water (HUA diet) for 16 weeks, as high-fat and high-fructose diets are reported to stimulate the overproduction of UA in systemic circulation, leading to the development of HUA in experimental animals [21]. Then, the HUA group mice were further randomly divided into three groups (n=10 each group): the HUA group was continue fed HUA diet; the HUA+Benz group was given HUA diet with intragastric administration of 20 mg/kg benzbromarone (a uricosuric agent clinically used for the regulation of HUA) for 4 weeks; and the HUA+Met group was given HUA diet with intragastric administration of 300 mg/kg metformin for 4 weeks.

Glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed at the end of experiment. On the second day, all mice were killed to obtain adipose tissue and serum for a trial scheduled. Euthanasia was done under deep anaesthesia using 5% isoflurane inhalation and maintained throughout the surgical procedure.

Culture and differentiation of 3T3-L1 cells

The 3T3-L1 pre-adipocyte cells were purchased from the American Type Culture Collection (CL-173, Rockville, MD, U.S.A.), and cultured in DMEM (Invitrogen, Carlsbad, CA, U.S.A.) with 10% bovine calf serum (HyClone, Logan, UT, U.S.A.) at 37°C and 5% CO2. When grown to post-confluence for 48 h, cells were stimulated with differentiation mixture medium containing 10% FBS (Sciencell, Carlsbad, CA, U.S.A.), 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 5 μg/ml insulin (Sigma, St Louis, MO, U.S.A.) for 48 h. After that, cell medium was changed to DMEM containing 10% FBS and 5 μg/ml insulin for another 48 h, and then replaced with normal culture medium for 6-8 days [22]. Subsequently, these cells were incubated with UA for 24 h. Here, 4 mg/dl was set as norm uricemia (approximately 250 mol/l), 8 mg/dl as HUA (500 mol/l) and 12 mg/dl as severe HUA (750 mol/l), which is consistent with the clinical assessment.

Oil red O staining

To measure intracellular lipid accumulation, an Oil red O Test Kit (Nanjing Jiancheng Bioengineering Institute, China) was used. 3T3-L1 cells were fixed with 4% paraformaldehyde at room temperature for 1 h and then incubated with filtered Oil red O solution (Oil red O stock solution: dilution = 5:2) for 30 min. Finally, cells were incubated with haematoxylin for 5 min and washed three times with PBS [23]. A light microscope was used to observe the staining results, and images were obtained at 200× magnification.

Western blot analysis

Total protein from eWAT and 3T3-L1 cells was extracted using RIPA buffer containing 10% phosphatase inhibitor and 1% protease inhibitor. The samples were resolved in sodium dodecyl sulfate (SDS)–polyacrylamide gels and then transferred to polyvinylidene fluoride (PVDF) membranes. Next, membranes were blocked with 5% BSA and incubated overnight at 4°C with the following primary antibodies: CD36 (catalogue no. ab133625, Abcam, Cambridge, MA, U.S.A.); ACC (catalogue no. 3676, CST, Boston, MA, U.S.A.); FASN (catalogue no. 3180, CST, Boston, MA, U.S.A.); ATGL (catalogue no. ab109251, Abcam, Cambridge, MA, U.S.A.); HSL (catalogue no. 4107, CST, Boston, MA, U.S.A.); UCP1 (catalogue no. ab10983, Abcam, Cambridge, MA, U.S.A.); PGC1α (catalogue no. ab54481, Abcam, Cambridge, MA, U.S.A.); and PRDM16 (catalogue no. ab106410, Abcam, Cambridge, MA, U.S.A.). Then the membranes were incubated with secondary antibody (catalogue no. ZB-2301/2305, ZSGB-BIO, Beijing, China) after washing [24]. After that, the signals were incubated with ECL light reagent for 3 min (Beyotime, Shanghai, China). Lastly, the blots were imaged using a gel documentation system (BIO-RAD, Hercules, CA, USA), and images of blots were analysed by the Image Lab.

EdU proliferation assay

After PBS washing, the 3T3-L1 cells were incubated in 10 μmol/l EdU (RiboBio, Guangzhou, China) for 2 h. After that, the cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.5% Triton-X for 10 min. The number of cycling cells was detected by DNA staining [25]. Fluorescence microscopy was used for imaging.

Immunofluorescence staining

After PBS washing three times, 3T3-L1 cells were fixed with 4% pre-cooled paraformaldehyde for 20 min and permeabilized 10 min with 0.5% Triton-X-100. Then, the cells were blocked for 1 h with 5% BSA and incubated overnight at 4°C with anti-UCP1 (catalogue no. ab10983, Abcam, Cambridge, MA, U.S.A.). Following that, cells were incubated with CoraLite488–conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (catalogue no. SA00013-2, Proteintech, Rosemont, IL, U.S.A.) and stained with DAPI [26]. The expression of UCP1 was observed and photographed using fluorescence microscopy.

H&E staining

Paraffin sections were dewaxed to water, stained in haematoxylin solution for 5 min, and then differentiated by 70% hydrochloric alcohol for 10 s. Next, paraffin sections were stained in eosin solution for 60 s after washing with deionized water. The samples were then cleared in xylene after washing and dehydrating by graded ethanol. Finally, the sections were mounted in neutral balsam and images were obtained under a laser scanning microscope [27].

Immunohistochemistry staining

Paraffin-embedded eWAT sections were incubated overnight at 4°C with anti-F4/80 (catalogue no. 70076, CST, Boston, MA, U.S.A.) or UCP1 (catalogue no. ab10983, Abcam, Cambridge, MA, U.S.A.). Then the sections were incubated at 37°C for 30 min with peroxidase-conjugated goat anti-rabbit IgG (catalogue no. ZDR-5306, ZSGB-BIO, Beijing, China) [28]. The images were obtained under a laser scanning microscope.

mRNA sequencing

The 3T3-L1 cells were collected and sent to Novogene Corporation (Beijing, China) for mRNA sequencing. Briefly, 3 μg RNA from each sample was used for analysis. Sequencing libraries were generated and each sample were index coded and clustered on a cBot Cluster Generation System. After that, the library preparations were sequenced on an Illumina HiSeq platform, and 125 bp/150 bp paired-end reads were generated. In-house Perl scripts were used to process raw data in fastq format. Differential expression analysis of two conditions/groups was performed using the DESeq2 R package (1.16.1). Statistical enrichment of deferentially expressed genes in KEGG pathways was tested using the Cluster Profiler R package.

Biochemical analysis

The levels of serum triglyceride (TG), FFAs and UA were determined using assay kits (catalogue no. ETGA-200, EFFA-100, DIUA-250, BioAssay Systems, Hayward, CA, U.S.A.). The TG contents of 3T3-L1 and eWAT were determined using assay kits (catalogue no. E1013, Applygen, Beijing, China).

Glucose Tolerance Test (GTT)

Mice in each group received an intragastric administration of 50% glucose (5 ml/kg) after a 16-h fast. The sample blood glucose was detected from tail incision by a portable glucometer (Roche, USA) at baseline and 15, 30, 60, 90 and 120 min after glucose administration [29].

Insulin Tolerance Test (ITT)

Mice in each group were injected intraperitoneally with regular human insulin (1.5 U/kg) after a 6-h fast. The level of blood glucose of samples from tail incision was detected by a portable glucometer (Roche, USA) at baseline and 15, 30, 60, 90 and 120 min after insulin administration [29].

Statistical analyses

The data were presented as the means ± SEM. Differences between two groups were analysed by Student’s t test, and multiple-group comparisons were analysed using one-way ANOVA followed by Tukey’s tests. Figures were analysed using GraphPad Prism 7. A P value<0.05 was considered statistically significant.

Results

Adipocytes in patients with HUA disease showed hypertrophy and decreased UCP1 expression

H&E staining was performed to provide an overview of the specimen and to quantify the size of the adipocytes. As Figure 1A shows, the adipocyte size in patients with HUA was larger than that in the Non-HUA group, and many broken adipocytes could be observed in the HUA group. Moreover, immunostaining showed that the expression of UCP1 was lower in the HUA group (Figure 1B), indicating a reduction in beige-like characteristics in the adipose tissue of patients with HUA.

UA induces adipocytes hypertrophy through promoting lipogenesis and inhibiting lipolysis

Figure 1
UA induces adipocytes hypertrophy through promoting lipogenesis and inhibiting lipolysis

(A) H&E staining of adipose tissue from Non-HUA and HUA patients (n=8). (B) Immunohistochemical staining of UCP1 on adipose tissue paraffin section from Non-HUA and HUA patients (n=8). Magnification: 200×; scale bar: 50 μm (n=8). (C) Photographs for Oil Red O staining of 3T3-L1 cells treated with the increasing concentration of UA (0, 4, 8, 12 mg/dl) for 24 h (n=5). Magnification: 200×; scale bar: 200 μm. (D) Intracellular triglyceride levels normalized to cell protein (n=4). (E) Western blot detection and quantification of CD36, ACC and FASN protein level (n=4). (F) Western blot detection and quantification of HSL and ATGL protein level (n=4). Data are shown as mean ± SEM; *P<0.05, **P<0.01, ***P<0.001 vs. 0 group.

Figure 1
UA induces adipocytes hypertrophy through promoting lipogenesis and inhibiting lipolysis

(A) H&E staining of adipose tissue from Non-HUA and HUA patients (n=8). (B) Immunohistochemical staining of UCP1 on adipose tissue paraffin section from Non-HUA and HUA patients (n=8). Magnification: 200×; scale bar: 50 μm (n=8). (C) Photographs for Oil Red O staining of 3T3-L1 cells treated with the increasing concentration of UA (0, 4, 8, 12 mg/dl) for 24 h (n=5). Magnification: 200×; scale bar: 200 μm. (D) Intracellular triglyceride levels normalized to cell protein (n=4). (E) Western blot detection and quantification of CD36, ACC and FASN protein level (n=4). (F) Western blot detection and quantification of HSL and ATGL protein level (n=4). Data are shown as mean ± SEM; *P<0.05, **P<0.01, ***P<0.001 vs. 0 group.

UA induces adipocyte hypertrophy and inhibits the hyperplasia of pre-adipocytes

In order to determine the impact of UA on adipocyte’s morphology and function, we exposed 3T3-L1 adipocytes to gradually increasing concentrations of UA (from 0 to 12 mg/dl) and found a dose-dependent increase in lipid droplet volume (Figure 1C) and intracellular triglyceride content (Figure 1D) in 3T3-L1 cells. To illuminate the molecular mechanism of how UA stimulates fat synthesis and induces adipocyte hypertrophy, specific proteins involved in lipogenesis and lipolysis were measured. As shown in Figure 1E, the expression of fatty acid translocase CD36 and the lipogenesis-related proteins ACC and FASN was dose-dependently increased, while the primary lipases responsible for lipolysis, including hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), decreased with increasing UA concentration (Figure 1F). Together, these findings suggested that UA promoted lipogenesis and inhibited lipolysis by regulating the expression of related proteins, which then induced the accumulation of TGs in adipocytes, leading to cell hypertrophy.

Moreover, UA inhibited the hyperplasia of pre-adipocytes, manifesting as number of EdU-positive cells decreased as the concentration of UA increased (Figure 2A). The increased number of hypertrophic adipocytes and impairment of small adipocyte proliferation together are responsible for the metabolic inflexibility of adipose tissue.

UA inhibits hyperplasia and beige adipocytes formation in 3T3-L1cells

Figure 2
UA inhibits hyperplasia and beige adipocytes formation in 3T3-L1cells

(A) EdU staining of 3T3-L1 pre-adipocytes and quantitative analyses of the numbers of EdU-positive cells (n=5). Magnification: 100×; scale bar: 400 μm. Red: EdU staining; Blue: DAPI staining for nucleus. (B) Fluorescence intensity detection of UCP1 in 3T3-L1 cells treated with increasing concentration of UA and quantitative analyses of UCP1-positive cells (n=5). Magnification: 200×; scale bar: 200 μm. (C) Western blot detection and quantification of UCP1, PGC1α and PRDM16 protein levels (n=4). Data are shown as mean ± SEM; *P<0.05, **P<0.01, ***P<0.001 vs. 0 group.

Figure 2
UA inhibits hyperplasia and beige adipocytes formation in 3T3-L1cells

(A) EdU staining of 3T3-L1 pre-adipocytes and quantitative analyses of the numbers of EdU-positive cells (n=5). Magnification: 100×; scale bar: 400 μm. Red: EdU staining; Blue: DAPI staining for nucleus. (B) Fluorescence intensity detection of UCP1 in 3T3-L1 cells treated with increasing concentration of UA and quantitative analyses of UCP1-positive cells (n=5). Magnification: 200×; scale bar: 200 μm. (C) Western blot detection and quantification of UCP1, PGC1α and PRDM16 protein levels (n=4). Data are shown as mean ± SEM; *P<0.05, **P<0.01, ***P<0.001 vs. 0 group.

UA reduces the beige-like characteristics of adipocytes

Subsequently, we measured the effects of UA on the beiging process of adipocytes. We found that with increasing UA concentration, the fluorescence intensity of UCP1 was decreased (Figure 2B) and that the expression of the main regulators of beige adipocyte formation, such as UCP1, PGC1α and PRDM16, was also significantly reduced (Figure 2C). To further determine whether β-adrenergic agonist-induced browning is also inhibited by UA, adipocytes were given the β-adrenergic agonist CL316,243. As Supplementary Figure S1B shows, the expression of UCP1, PGC1α and PRDM16 was inhibited by UA according to Western blot analysis, and the extent of UCP1 in the HUA+CL group was also less than that in the control+CL group, as shown by fluorescence intensity (Supplementary Figure S1C). These results indicated that beige adipocyte formation was suppressed by UA regardless of whether it was stimulated by a β-adrenergic agonist.

Adipocytes express URAT1, which is a key mediator transporting UA to cells [15], and previous study has found that expression of URAT1 gene in HUA population was associated with serum UA level [30]. Here, we also evaluated URAT1 expression in adipocyte to check if it is the key factor in regulating adipocyte function by UA. Our results showed that the expression of URAT1 didn’t have a strong correlation with UA dosage (Supplementary Figure S1A).

Leptin plays a key role in the UA-induced FFA metabolic disorder of adipocytes

To characterize the molecular mechanism of UA in adipocyte metabolism, mRNA-sequencing analysis was performed. A total of 1204 mRNAs were differentially expressed, including 658 up-regulated and 546 down-regulated mRNAs (Figure 3A). Among these differentially expressed mRNAs, leptin was found to be significantly down-regulated with the highest mean ratio according to heat map representations (Figure 3B). The KEGG analysis revealed that the ‘fatty acid metabolism’ pathway was highly enriched with differentially expressed mRNAs (Figure 3C), and leptin is a critical one among the mRNAs enriched in the fatty acid metabolism signalling pathway (Figure 3D). Moreover, among the leptin-related biological pathways, the ‘AMPK signalling pathway’ was at the forefront (Figure 3E). Therefore, we suggest that leptin is a key regulator in the UA-induced FFA metabolic disorder of adipocytes and that AMPK might be the main target of leptin.

Leptin plays a crucial role in UA-induced fatty acid metabolic disorder in adipocytes

Figure 3
Leptin plays a crucial role in UA-induced fatty acid metabolic disorder in adipocytes

(A) Volcano plot of transcriptome in 3T3-L1 cells treated with or without UA (n=3). Among 1204 genes, 658 were up-regulated, while 546 were down-regulated; green: down-regulated; red: up-regulated. (B) Heatmap of genes treated with or without UA, and along with the top affected gene-leptin which was highlight in red rectangle. blue: down-regulated; red: up-regulated. (C) KEGG analysis demonstrated ‘fatty acid metabolism’ was a major enriched alteration in complex cellular pathways. ‘Fatty acid metabolism’ was highlight in red color. (D) KEGG pathway analysis showed leptin was the mainly targeted gene in UA-induced fatty acid metabolic disorder in adipocytes. The color depth determined the fold of mRNA changes. Big red ellipse: fatty acid metabolism; small red ellipse: leptin. (E) KEGG pathway analysis showed among the leptin-related biological pathways, ‘AMPK signaling pathway’ was at the front line. Red color: AMPK signaling pathway. KEGG: Kyoto encyclopedia of Genes and Genomes; Lep: leptin.

Figure 3
Leptin plays a crucial role in UA-induced fatty acid metabolic disorder in adipocytes

(A) Volcano plot of transcriptome in 3T3-L1 cells treated with or without UA (n=3). Among 1204 genes, 658 were up-regulated, while 546 were down-regulated; green: down-regulated; red: up-regulated. (B) Heatmap of genes treated with or without UA, and along with the top affected gene-leptin which was highlight in red rectangle. blue: down-regulated; red: up-regulated. (C) KEGG analysis demonstrated ‘fatty acid metabolism’ was a major enriched alteration in complex cellular pathways. ‘Fatty acid metabolism’ was highlight in red color. (D) KEGG pathway analysis showed leptin was the mainly targeted gene in UA-induced fatty acid metabolic disorder in adipocytes. The color depth determined the fold of mRNA changes. Big red ellipse: fatty acid metabolism; small red ellipse: leptin. (E) KEGG pathway analysis showed among the leptin-related biological pathways, ‘AMPK signaling pathway’ was at the front line. Red color: AMPK signaling pathway. KEGG: Kyoto encyclopedia of Genes and Genomes; Lep: leptin.

Leptin alleviates the UA-induced metabolic dysfunction of adipocytes

To verify the hypothesis that leptin is a key regulator in the UA-induced FFA metabolic disorder of adipocytes, we tested the leptin levels in 3T3-L1 cells treated with UA (Figure 4B) and epididymal white adipose tissue (eWAT) of HUA mice (Figure 4C) and found that they were both significantly decreased, which was consistent with the mRNA-sequencing results (Figure 4A). Then, we added leptin to the culture medium of UA-treated 3T3-L1 cells and found that leptin decreased the expression of the lipogenesis-related proteins CD36, ACC and FASN (Supplementary Figure S2A) while increasing the lipolysis regulators ATGL and HSL (Supplementary Figure S2B), which led to a reduction in lipid droplet volume (Supplementary Figure S2C) and triglyceride accumulation in adipocytes (Supplementary Figure S2D). Moreover, leptin reversed the hyperplasia reduction of adipocytes caused by UA, manifested as an increased number of EdU-positive cells (Supplementary Figure S3A).

UA induces adipocytes hypertrophy via inhibiting leptin-AMPK signaling pathway in 3T3-L1 cells

Figure 4
UA induces adipocytes hypertrophy via inhibiting leptin-AMPK signaling pathway in 3T3-L1 cells

(A) Fold changes of leptin in the mRNA microarray analysis (n=3). (B) Concentration of leptin in 3T3-L1 cells treated with or without UA (n=4). (C) Concentration of leptin in isolated adipocytes of eWAT from control and HUA mice (n=7). (D) Western blot detection and quantification of relative pAMPK/AMPK ratio in four groups with increasing concentration of UA (n=4); *P<0.05, **P<0.01, ***P<0.001 vs. 0 group. (E) Western blot detection and quantification of relative pAMPK/AMPK ratio in normal, HUA and HUA+Lep groups (n=4). (F) Western blot detection and quantification of CD36, ACC and FASN protein levels (n=4). (G) Western blot detection and quantification of HSL and ATGL protein levels (n=4). (H) Photographs for Oil Red O staining (n=5). Magnification: 200×; scale bar: 200 μm. (I) Intracellular triglyceride levels normalized to cell protein (n=4). Met: Metformin; AICAR: 5-aminoimidazole-4-carboxamide ribonucleotide. Data are shown as mean ± SEM; *P<0.05, **P<0.01, ***P<0.001 vs. Control group, §P<0.05, §§P<0.01, §§§P<0.001 vs. HUA group.

Figure 4
UA induces adipocytes hypertrophy via inhibiting leptin-AMPK signaling pathway in 3T3-L1 cells

(A) Fold changes of leptin in the mRNA microarray analysis (n=3). (B) Concentration of leptin in 3T3-L1 cells treated with or without UA (n=4). (C) Concentration of leptin in isolated adipocytes of eWAT from control and HUA mice (n=7). (D) Western blot detection and quantification of relative pAMPK/AMPK ratio in four groups with increasing concentration of UA (n=4); *P<0.05, **P<0.01, ***P<0.001 vs. 0 group. (E) Western blot detection and quantification of relative pAMPK/AMPK ratio in normal, HUA and HUA+Lep groups (n=4). (F) Western blot detection and quantification of CD36, ACC and FASN protein levels (n=4). (G) Western blot detection and quantification of HSL and ATGL protein levels (n=4). (H) Photographs for Oil Red O staining (n=5). Magnification: 200×; scale bar: 200 μm. (I) Intracellular triglyceride levels normalized to cell protein (n=4). Met: Metformin; AICAR: 5-aminoimidazole-4-carboxamide ribonucleotide. Data are shown as mean ± SEM; *P<0.05, **P<0.01, ***P<0.001 vs. Control group, §P<0.05, §§P<0.01, §§§P<0.001 vs. HUA group.

In addition, leptin restored the beige adipocyte formation inhibited by UA, as shown by the enhanced fluorescence intensity of UCP1 (Supplementary Figure S3B). Furthermore, the expression of the main regulators of beige adipocyte activity, including UCP1, PGC1α and PRDM16, was also up-regulated after leptin administration in 3T3-L1 cells (Supplementary Figure S3C). These results suggest that leptin plays a key role in the UA-induced metabolic dysfunction of adipocytes.

Metformin protects against UA-induced cell hypertrophy, hyperplasia inhibition and suppression of beige-like characteristics via AMPK activation

To further dissect the molecular mechanism of UA-induced metabolic dysfunction in adipocytes, we tried to determine the downstream target of leptin. According to the bioinformatics analysis, we hypothesized that leptin interacted with AMPK in UA-treated adipocytes. Then, we tested the p-AMPK/AMPK ratio in 3T3-L1 cells and found that the expression ratio decreased along with the increase in UA concentration (Figure 4D), and this can be reversed by the addition of leptin (Figure 4E). These results indicated that UA inhibited the phosphorylation of AMPK by reducing the expression of leptin.

To investigate the role of AMPK in UA-induced adipose tissue dysfunction, 3T3-L1 cells were given two AMPK activators, namely, metformin and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). AMPK activation rebalanced FFA metabolism in adipocytes by decreasing the expression of the lipogenesis-related proteins CD36, ACC and FASN (Figure 4F) and increasing the lipolysis regulators ATGL and HSL (Figure 4G), which protected 3T3-L1 cells from UA-induced hypertrophy and excessive TG accumulation, as reflected by the decreased volume of lipid droplets (Figure 4H) and cellular TG content (Figure 4I). AMPK activation also restored the hyperplasia of adipocytes (Figure 5A) and beige adipocyte formation that was suppressed by UA (Figure 5B,C). Taken together, these findings demonstrated that metformin protects adipocytes from UA-induced cell hypertrophy and hyperplasia inhibition and helps adipocytes restore beige-like characteristics by activating AMPK.

UA depresses hyperplasia and beige adipocytes formation via inhibiting leptin-AMPK signaling pathway in 3T3-L1 cells

Figure 5
UA depresses hyperplasia and beige adipocytes formation via inhibiting leptin-AMPK signaling pathway in 3T3-L1 cells

(A) EdU staining of 3T3-L1 pre-adipocytes and quantitative analyses of the numbers of EdU+ cells (n=5). Magnification: 100×; scale bar: 400 μm. Red: EdU staining; Blue: DAPI staining for nucleus. (B) Fluorescence intensity detection of UCP1 in 3T3-L1 cells treated with normal, HUA, HUA+Met, HUA+AICAR for 24 h and quantitative analyses of UCP1+ cells (n=5). Magnification: 200×; scale bar: 200 μm. (C) Western blot detection and quantification of UCP1, PGC1α and PRDM16 protein levels in 3T3-L1 cells (n=4). Data are shown as mean ± SEM; ***P<0.001 vs. Control group, §P<0.05, §§P<0.01, §§§P<0.001 vs. HUA group.

Figure 5
UA depresses hyperplasia and beige adipocytes formation via inhibiting leptin-AMPK signaling pathway in 3T3-L1 cells

(A) EdU staining of 3T3-L1 pre-adipocytes and quantitative analyses of the numbers of EdU+ cells (n=5). Magnification: 100×; scale bar: 400 μm. Red: EdU staining; Blue: DAPI staining for nucleus. (B) Fluorescence intensity detection of UCP1 in 3T3-L1 cells treated with normal, HUA, HUA+Met, HUA+AICAR for 24 h and quantitative analyses of UCP1+ cells (n=5). Magnification: 200×; scale bar: 200 μm. (C) Western blot detection and quantification of UCP1, PGC1α and PRDM16 protein levels in 3T3-L1 cells (n=4). Data are shown as mean ± SEM; ***P<0.001 vs. Control group, §P<0.05, §§P<0.01, §§§P<0.001 vs. HUA group.

Metformin alleviates UA-induced serum FFA elevation and insulin resistance in HUA mice

We also evaluated the effects of benzbromarone (a uricosuric agent) and metformin on improving adipose tissue function in mice. The results were consistent with those in vitro. Both benzbromarone and metformin significantly reduced the UA-stimulated intake of fatty acids and lipogenesis of adipocytes, as evidenced by the decreased expression of CD36, ACC and FASN compared with the HUA group (Figure 6A), and reversed the inhibitory effect of UA on lipolysis with the increased expression of HSL and ATGL (Figure 6B). Histologically, both benzbromarone and metformin alleviated the hypertrophic features of adipocytes, with a switch toward smaller adipocytes (Figure 6C,D). Moreover, they decreased triglyceride accumulation in the eWAT of HUA mice (Figure 6E) and increased the number of adipocytes by facilitating hyperplasia (Figure 6F). Additionally, UA-induced macrophage aggregation around eWAT was alleviated by benzbromarone and metformin, as shown by F4/80 (macrophage) antibody staining (Figure 6G).

Metformin alleviates HUA-induced adipocytes hypertrophy and hyperplasia inhibition in eWAT of mice

Figure 6
Metformin alleviates HUA-induced adipocytes hypertrophy and hyperplasia inhibition in eWAT of mice

(A) Western blot detection and quantification of CD36, ACC and FASN protein levels of eWAT of mice (n=5). (B) Western blot detection and quantification of HSL and ATGL protein levels of eWAT of mice (n=5). (C) H&E staining of eWAT from each group for the diagnosis of adipocyte size (n=5). (D) Adipocyte size distribution in eWAT of mice (n=5 mice per group). (E) Triglyceride levels in eWAT normalized to cell protein (n=9). (F) Relative adipocyte number in eWAT of mice (n=5 mice per group). (G) Immunohistochemical staining of F4/80 on eWAT paraffin section (n=5). Magnification: 200×; scale bar: 200 μm. Benz: benzbromarone; Met: Metformin. Data are shown as mean ± SEM; ***P<0.001 vs. Control group, §P<0.05, §§P<0.01, §§§P<0.001 vs. HUA group.

Figure 6
Metformin alleviates HUA-induced adipocytes hypertrophy and hyperplasia inhibition in eWAT of mice

(A) Western blot detection and quantification of CD36, ACC and FASN protein levels of eWAT of mice (n=5). (B) Western blot detection and quantification of HSL and ATGL protein levels of eWAT of mice (n=5). (C) H&E staining of eWAT from each group for the diagnosis of adipocyte size (n=5). (D) Adipocyte size distribution in eWAT of mice (n=5 mice per group). (E) Triglyceride levels in eWAT normalized to cell protein (n=9). (F) Relative adipocyte number in eWAT of mice (n=5 mice per group). (G) Immunohistochemical staining of F4/80 on eWAT paraffin section (n=5). Magnification: 200×; scale bar: 200 μm. Benz: benzbromarone; Met: Metformin. Data are shown as mean ± SEM; ***P<0.001 vs. Control group, §P<0.05, §§P<0.01, §§§P<0.001 vs. HUA group.

In addition, UCP1 staining of eWAT showed that the expression of UCP1 in the HUA group was significantly lower compared with the control group, while benzbromarone and metformin increased the UCP1 expression in eWAT (Figure 7A). Consistently, the main proteins regulating adipose browning, namely, UCP1, PGC1α and PRDM16, were also significantly up-regulated in mice from the benzbromarone and metformin treatment groups (Figure 7B).

Metformin decreases level of serum UA, FFA and TG, and alleviates insulin resistance

Figure 7
Metformin decreases level of serum UA, FFA and TG, and alleviates insulin resistance

(A) Immunohistochemical staining of UCP1 on eWAT paraffin section (n=5). Magnification: 200×; scale bar: 200 μm. (B) Western blot detection and quantification of UCP1, PGC1α and PRDM16 protein levels in eWAT (n=5). (C) Serum levels of uric acid. (D) Serum levels of FFA. (E) Serum levels of TG. (F) Glucose tolerance test (GTT). (G) Insulin tolerance test (ITT) (n=8). Data are shown as mean ± SEM; *P<0.05, **P<0.01, ***P<0.001 vs. Control group, §P<0.05, §§P<0.01, §§§P < 0.001 vs. HUA group. (H) A proposed schematic diagram for the effect of UA on adipocyte. UA inhibits phosphorylation of AMPK by reducing the expression of leptin.

Figure 7
Metformin decreases level of serum UA, FFA and TG, and alleviates insulin resistance

(A) Immunohistochemical staining of UCP1 on eWAT paraffin section (n=5). Magnification: 200×; scale bar: 200 μm. (B) Western blot detection and quantification of UCP1, PGC1α and PRDM16 protein levels in eWAT (n=5). (C) Serum levels of uric acid. (D) Serum levels of FFA. (E) Serum levels of TG. (F) Glucose tolerance test (GTT). (G) Insulin tolerance test (ITT) (n=8). Data are shown as mean ± SEM; *P<0.05, **P<0.01, ***P<0.001 vs. Control group, §P<0.05, §§P<0.01, §§§P < 0.001 vs. HUA group. (H) A proposed schematic diagram for the effect of UA on adipocyte. UA inhibits phosphorylation of AMPK by reducing the expression of leptin.

In serum, both benzbromarone and metformin treatment decreased the concentrations of FFAs and TG (Figure 7D,E), enhanced glucose clearance and improved insulin sensitivity (Figure 7F,G) in HUA mice. Moreover, the level of UA also decreased after metformin treatment (Figure 7C). Overall, we confirmed that metformin preserved the function of adipose tissue by decreasing adipocyte hypotrophy and increasing adipose beiging and consequently stimulated its favourable FFA metabolic flexibility. Therefore, metformin attenuated HUA-induced serum FFA elevation and insulin resistance.

Discussion

In the present study, we demonstrated that UA directly induced adipocyte hypertrophy and inhibited the hyperplasia of pre-adipocytes. Moreover, UA decreased the formation of beige adipocytes. The main mechanism was through leptin-AMPK pathway inhibition. These alterations damaged the capability of FFA metabolism in adipose tissue, which further induced elevation of serum FFAs and TG and caused insulin resistance. These abnormal metabolic processes can be reversed after AMPK activation by metformin.

Clinical trials have shown that serum UA levels are closely related to increased serum TG and FFA levels [7]. Similarly, in animal experiments, serum TG and FFA levels increased significantly after purine-rich diet-induced serum UA elevation [31]. Elevated FFAs may be taken up and ectopically deposited in pancreatic islets, disturbing the function of β cells and impairing insulin secretion. Moreover, excess serum FFAs impair hippocampal insulin signalling and induce insulin resistance [32]. However, the exact mechanism by which UA elevates serum FFAs remains unclear. A high-fat and high-fructose diet-induced HUA model is good for simulating the growth and development process of HUA disease, as it has been reported that obese individuals always have a higher UA level [16].

WAT is a vital organ in maintaining whole-body energy homeostasis [33]. It stores excess energy in the form of TG while releasing FFAs and glycerol by lipolysis upon various energy demands [34]. Under conditions of constant lipid accumulation, adipocytes become hypertrophic and then hyperplastic to generate new adipocytes, so as to increase lipid storage capacity and keep circulating FFAs and blood glucose below toxic levels [35]. Once lipid accumulation exceeds the adipocyte’s storage capacity, the cell will cease to take up or store more FFAs [36]. Moreover, over-hypertrophied adipocytes could cause hypoxia and cell death, resulting in a large and uncontrolled release of FFAs [37], which further induces serum FFA level elevation and macrophage infiltration around adipocytes [38]. An increase in macrophage number has been found following the rising levels of FFAs and cholesterol [39]. In the present study, we found that UA directly increased the expression of fatty acid translocase CD36, as well as the lipogenesis-related proteins ACC and FASN, while decreased the expression of lipolysis-related proteins HSL and ATGL, which led to excessive lipid accumulation in adipocytes that facilitated cell hypertrophy. On the other hand, UA inhibited adipose tissue generation of more functional cells through hyperplasia, causing the mismatch between lipid storage demands and storage space. As a result, serum FFAs and lipids increased and accumulated in other non-adipose tissues; therefore, lipotoxic insults occurred. This is consistent with previous research showing that the metabolic inflexibility of adipose tissue is associated with metabolic diseases [40].

WAT also contains beige adipocytes that are transdifferentiated from white adipocytes under external stimulation and possess a brown-like feature. The accumulation of beige adipocytes in WAT is called ´browning’ [41]. It has been found that adipose browning could absorb circulating exogenous FFAs and glucose as fuel to generate heat, helping to improve metabolic health [42,43]. In contrast, losing active beige adipocytes or decreasing the browning capacity of adipose tissue results in a reduction in energy expenditure and contributes to progressive metabolic decline [44,45]. In the present study, we found that a high level of UA could suppress beige adipocyte formation by inhibiting the expression of UCP1, PGC1a and PRDM16. Therefore, we suggested that UA decreased the FFA uptake and utilization rate of adipose tissue by inhibiting beige adipocyte formation.

URAT1 is a surface protein responsible for transporting of UA into cells [46,47]. Previous studies showed that the expression of URAT1 gene in HUA population was positively associated with serum UA level [30], and inhibition of the expression of URAT1 in cells can alleviate UA-induced cellular dysfunction [48]. Here, we wanted to study whether high UA level could influence adipocyte metabolism through upregulating the expression of URAT1 protein. Our results showed that the expression of URAT1 in adipocytes did not have a strong correlation with UA dosage, which implies that URAT1 is not a key target for UA-induced adipocyte metabolic disorders.

To further determine the impact of UA on adipocyte’s morphology and function, mRNA-sequencing analysis was performed, and we found that leptin mRNA decreased sharply in 3T3-L1 cells treated with UA and was closely related to FFA metabolism according to mRNA microarray and KEGG enrichment pathway analysis. In 3T3-L1 cells, after leptin administration, UA-induced FFA metabolic disorders were prevented, which means that leptin is the functional target of UA.

As leptin therapy is not ideal for patients with hyperleptinemia who show poor responses to exogenous leptin [49,50], we tried to identify the downstream target of leptin, which could be an alternative therapeutic strategy to eliminate metabolic disorders induced by HUA. KEGG enrichment pathway and bioinformatics analysis indicated that leptin was associated with AMPK and its related signalling pathway. Other studies also showed that leptin could directly stimulate AMPK to reduce ACC activity, which decreases FFA synthesis [31] and increases the oxidation of FFAs [51]. Targeting the AMPK pathway could bypass leptin resistance and therefore represents an potential treatment method for metabolic disease [52]. In our study, we found that as the concentration of UA increased, both leptin and AMPK phosphorylation decreased, and leptin administration ameliorated UA-induced AMPK phosphorylation inhibition, which suggested that leptin–AMPK pathway inhibition existed in UA-affected adipose tissue. Moreover, AMPK activation by metformin also reversed UA-induced adipocyte hypertrophy and inhibited beige adipocyte formation both in vitro and in vivo. After metformin treatment, the levels of serum FFAs and TG were all reduced, and insulin sensitivity and impaired glucose metabolism were also improved. All these results proved the concept that the leptin–AMPK pathway plays a key role in UA-induced adipose tissue dysfunction.

Metformin has been used widely in the treating Type 2 diabetes. In addition to its hypoglycaemic effect, the drug can also improve other metabolic processes. A study showed that metformin could suppress abnormal extracellular matrix remodelling by repressing TGF-β1-induced fibrogenesis in adipose tissue and ameliorate insulin resistance in obesity via AMPK activation [53]. In addition, Miguel A et al. suggested that metformin-induced AMPK activation can also enhance fat oxidation and reduce lipogenesis in sucrose-fed rat hepatocytes [20]. In line with their findings, we found that metformin could alleviate HUA-induced serum FFA elevation and insulin resistance by improving adipose tissue function. Our results suggest that metformin might be an alternative treatment strategy for HUA-related metabolic disorders, especially for patients with HUA and diabetes mellitus.

Conclusion

In summary, our study elucidated the direct effect of UA on adipose tissue and proved that UA damaged the metabolic and beiging function of adipocytes by inhibiting the leptin-AMPK pathway (Figure 7H). These changes resulted in the dysfunction of adipose metabolism, which led to elevation of serum FFAs and TG, as well as insulin resistance. Our results suggested that metformin could be a novel therapeutic agent for HUA-related glycolipid metabolic disorder.

Clinical perspectives

  • Hyperuricaemia (HUA) significantly increases the risk of metabolic syndrome and is strongly associated with the increased prevalence of high serum free fatty acids (FFAs) and insulin resistance. However, the detailed mechanism remains unclear.

  • Our examinations of white adipose tissue (WAT) from normal and patients with HUA disease revealed that WAT in patients with HUA showed hypertrophy and decreased UCP1 expression. To study the major mechanism, we built both in vitro and in vivo models of HUA and found that high levels of UA induced adipocyte hypertrophy, inhibition of hyperplasia and a reduction in beige-like characteristics by inhibiting the leptin-AMPK pathway, which damaged the FFA metabolic function of adipose tissue and induced metabolic disorders. Moreover, metformin-induced activation of AMPK expression significantly attenuated UA-induced FFA metabolism impairment, which subsequently alleviated serum FFA elevation and insulin resistance in HUA mice.

  • Our findings suggest that metformin could become a novel potential treatment strategy for HUA-related metabolic disorders, especially for patients with HUA and diabetes mellitus.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This project was supported by grants from National Nature Scientific Foundation of China [grant numbers 81830012, 81670297 and 81800332]; Doctoral Fund of Ministry of Education of Heilongjiang Province [grant number LBH-Z17180]; and Harbin Medical University Science Innovation Foundation [grant number YJSKYCX2018-91HYD] also provided additional support.

Author Contribution

M.Q.S. and L.S. together conceived the study, designed the experiment, analyzed the datasets and wrote the manuscript. W.P.L., H.L., Y.W., Y.L. and Y.Y. conducted the experiments. S.L.Y., L.Q.Z., C.G.D. and C.Y.Z. analyzed and interpreted data. Z.W.P. designed experiments, interpreted data and edited the manuscript. Y.L. identified and formulated the research, supervised all experiments and gave final approval for the version to be published.

Acknowledgements

The authors thank Roberto Patarca (Johnson&Johnson Medical China) and Longfeng Rao (Institute for Biomechanics, Swiss Federal Institute of Technology, ETHz) for editing and drafting of the manuscript.

Abbreviations

     
  • AICAR

    5-aminoimidazole-4-carboxamide ribonucleotide

  •  
  • AMPK

    AMP-activated protein kinase

  •  
  • FFA

    free fatty acid

  •  
  • GTT

    glucose tolerance test

  •  
  • HUA

    hyperuricaemia

  •  
  • ITT

    insulin tolerance test

  •  
  • MCP-1

    monocyte chemotactic protein-1

  •  
  • TG

    triglyceride

  •  
  • UA

    uric acid

  •  
  • UCP1

    uncoupling protein 1

  •  
  • URAT1

    urate-anion exchanger 1

  •  
  • WAT

    white adipose tissue

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Author notes

*

These authors contributed equally to this work.

Supplementary data