FGF21 ameliorates diabetic cardiomyopathy by activating the AMPK-paraoxonase 1 signaling axis in mice

The prevalence of diabetes mellitus is growing rapidly. Amongst all the diabetic complications, cardiovascular disease is recognized as the primary cause of mortality in diabetic patients. Diabetic cardiomyopathy (DCM), a rare complication caused by diabetes and characterized by impaired cardiac structure and function, is not fatal but does inevitably progress to heart failure. A number of studies have indicated that DCM is considered to be associated with oxidative stress and mitochondrial dysfunction (which are regarded as the main incentives to initiate ventricular remodeling), and is characterized by fibrosis and heart dysfunction [1–3]. Therefore, the beneficial effects of attenuating diabetes-induced oxidative damage and the subsequent cardiac hypertrophy and fibrosis, are expected to prevent diabetes-induced cardiac injury and may be a potential therapeutic strategy for DCM.

Fibroblast growth factor 21 (FGF21), a novel hormone predominantly secreted by hepatic tissues, plays an important role in whole-body metabolic regulation during physiological and pathological conditions [4–7]. Mounting evidence from human studies indicates that several types of cardiovascular diseases including atherosclerosis, coronary heart disease, and myocardial infarction are associated with an elevated circulating FGF21 level [8,9]. Animal-based studies also demonstrate that FGF21 deficiency accelerates the development of these cardiovascular diseases in relevant animal models [10,11]. In ApoE-knockout mice, FGF21 ablation strongly accelerates the progression of atherosclerosis, and treatment with recombinant mouse FGF21 (rmFGF21) protein significantly attenuates atherosclerosis status [11]. Furthermore, FGF21 deficiency also accelerates isoproterenol- and ischemia–reperfusion-induced myocardial injury in mice [12–14], suggesting that FGF21 presents a cardiovascular protective effect in pathological status. Recently, our colleagues revealed that streptozotocin (STZ)-induced type 1 diabetic mice with FGF21 ablation were more susceptible to developing diabetic cardiomyopathy [15]. In addition, FGF21 deletion results in upregulated Nrf2-driven CD36 expression, exacerbated cardiac lipid uptake and accumulation, which in turn impairs cardiac lipid and glucose utilization, cardiac energy balance, as well as aggravates cardiac oxidative stress, and eventually accelerating the development of DCM [15]. However, the underlying mechanism by which FGF21 counteracts diabetes-induced myocardial injury remains enigmatic. In the present study, we use FGF21 KO mice and cellular experiments to explore the exact mechanism that FGF21 protects against DCM.

FGF21 KO mice in C57BL/6J background were generated as previously described [16]. All the mice were housed in a room with controlled temperature (23 ± 1°C), a 12-h light-dark cycle, and had free access to water and diet. For the STZ-induced diabetic model, mice were first fed with a high-fat diet (HFD) for 4 weeks and then received intraperitoneal injection of a single dosage of STZ (Sigma–Aldrich, St. Louis, MO, dissolved in 0.1 M sodium citrate buffer, pH 4.5) at 100 mg/kg body weight, while age-matched control mice were fed with HFD accordingly and received a single injection of the same volume of sodium citrate buffer. Five days after the last injection of STZ, mice with hyperglycemia (3 h fasting blood glucose levels >250 mg/dl) were defined as diabetic as described previously [17]. When the diabetic models had been successfully induced, FGF21 KO male mice were also intravenously injected with 1 × 10¹² viral particles of adeno-associated virus (AAV) encoding FGF21 or GFP. Glucose and insulin tolerance tests were performed as described previously [18]. All the animal studies were approved by the Animal Research Ethics Committee of Wenzhou Medical University.

Heart function was evaluated by transthoracic echocardiography (ECHO). ECHO was performed on mice using a Visual Sonics Vevo 2100 high-resolution imaging system (Visual Sonics, Toronto, ON, Canada) as described previously [19]. 2D and M-model images were obtained for measurements of systolic function [19], and diastolic function was assessed using pulsed-wave Doppler imaging as described in a previous study [20].

Plasma lipid profiles including total triglycerides, and total cholesterol levels were measured with commercial kits from Sigma (St. Louis, MO). Plasma levels of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein 1 (MCP-1) were analyzed with immunoassays from R&D System Inc. (Minneapolis, MN). Serum insulin levels were tested with immunoassays from the Antibody and Immunoassay Services at the University of Hong Kong.

Cardiac sections were stained with hematoxylin-eosin (H&E) for morphological analysis or stained with dihydroethidium (DHE) to determine superoxide production in heart tissues [21], and stained with Sirius Red to reflect collagen accumulation [15]. Cardiac sections were also used to determine the apoptosis of cardiomyocytes using an in situ cell death detection assay (Roche, Mannheim, Germany). All slides were examined using an Olympus biological microscope BX41, and images were captured using Olympus DP72 color digital camera.

Paraoxonase 1 (PON1) arylesterase activity and paraoxonase activity were determined as previously described [22,23]. In brief, PON1 arylesterase activity was spectrophotometrically measured using phenyl acetate as the substrate in 50-fold diluted serum (final) at 270 nm and 25°C. The assay mixture included stock buffer (500 mM phenyl acetate in methanol, 250-fold diluted) and reaction buffer (50 mM Tris/HCl, 1 mM CaCl₂, pH 8.0). The E₂₇₀ for the reaction is 1310 mol/l⁻¹·cm⁻¹ and 1 unit of arylesterase activity is equal to 1 μmol of phenyl acetate hydrolyzed per ml per min.

Paraoxonase activity was measured spectrophotometrically at 412 nm using paraoxon as the substrate in 40-fold diluted serum (final) at 25°C. The assay mixture included stock buffer (400 mM phenyl paraoxon in methanol, 100-fold diluted) and reaction buffer (1.0 mM CaCl₂ in 0.05 M glycine buffer, pH 10.5, and no additional NaCl). An extinction coefficient (at 412 nm) of 16900 mol/l⁻¹·cm⁻¹ was used for calculating units of paraoxonase activity, which is expressed as nanomoles of p-nitrophenol produced per min per ml of serum.

Cardiomyocytes were isolated from neonatal mice as described previously [24]. Cells were suspended in DMEM with 10% FBS (FBS-DMEM), seeded on to six-well plates, cultured overnight in FBS-DMEM with 5.6 mmol/l glucose, infected with adenovirus with dominant-negative (DN)-AMP-activated protein kinase (AMPK) for 24 h, and then replaced with FBS-DMEM with 33 mmol/l glucose (high glucose, HG) and rmFGF21 protein for various time points, as described in the figure legends. Intracellular reactive oxygen species (ROS) levels in mouse primary cardiomyocytes were determined by 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) or a fluorescent probe, as described in [25]. The fluorescence intensity (relative fluorescence units) was measured at an excitation and emission wavelength of 485 and 530 nm, respectively. Cardiomyocyte apoptosis was determined by flow cytometry.

Recombinant adenovirus for the expression of a constitutively DN version of AMPKα was prepared as described previously [26]. Constitutively, DN-AMPKα carries a single mutation of the truncated form of the AMPK catalytic subunit α1 (Asp¹⁵⁷ replaced by alanine). PON1 siRNA and a scramble control were designed and confirmed by our primary experiments, and the sequences were as follows: PON1 siRNA, 5′-TCTGCCTAGCATCAACGATAT-3′; β-klotho siRNA, 5′-UGCGCAAGGUCUCCGGUACUA-3′; and scramble, 5′-CATAGCCAATATATTTCCAGAT-3′. Recombinant adenovirus encoding GFP was used as the control. Primary mouse cardiomyocytes were infected with these adenoviruses at 50 p.f.u/cell for 36 h prior to HG or/and FGF21 treatment.

Mice were killed under deep anesthesia, and the cardiac tissues were immediately collected for cardiac mitochondria isolation using the procedures described previously [27]. Cultured cardiomyocytes in specific experiments were also collected for mitochondria isolation using the same methods. Measurement of mitochondrial respiratory chain (MRC) complex activities was performed using the same previously described methods [28].

Intracellular ROS levels in mouse primary cardiomyocytes were determined using CM-H2DCFDA or a fluorescent probe as described in [28]. The fluorescence intensity (relative fluorescence units) was measured at an excitation and emission wavelength of 485 and 530 nm, respectively.

Immunoblotting was performed as described previously [11]. The targetted proteins were probed with primary antibodies against phospho-AMPK (Thr¹⁹²), AMPK, phospho-acetyl-CoA carboxylase (ACC) (Ser⁷⁹), ACC, PON1, and GAPDH (Cell Signaling Technology Company). The protein bands were visualized with ECL reagents (GE Healthcare, Uppsala, Sweden) and quantitated using the NIH ImageJ software.

Real-time PCR was performed as described previously [11]. Simply, total RNA was extracted from cardiac tissues, and cDNA was synthesized by reverse transcription with random hexamer primers. Quantitative real-time PCR was performed using a SYBR Green QPCR system (Qiagen) with specific primers (Supplementary Table S1). The level of target gene expression was normalized against the GAPDH gene.

All data were expressed as mean ± S.E.M. Datasets were analyzed for statistical significance using the non-parametric Mann–Whitney U-test, Kruskal–Wallis, or two-way ANOVA analysis when specified. In all statistical comparisons, a P-value <0.05 was used to indicate a statistically significant difference.

To explore the relationship between FGF21 and DCM, 8-week-old male WT mice were used to generate a STZ/HFD-induced diabetic model as described in the Methods section. Different from the previous report [15], administration of STZ/HFD significantly increased serum FGF21 levels in WT mice (Figure 1A). Furthermore, cardiac FGF21 mRNA and protein levels were also markedly elevated compared with the control group (Figure 1B,C), suggesting that elevated FGF21 in STZ/HFD-induced diabetic mice may be involved in the pathogenesis of DCM.

To further explore the molecular mechanism of FGF21 in protecting against DCM, 8-week-old male FGF21 KO mice and age- and gender-matched WT littermate controls were used to generate a STZ/HFD-induced diabetic model as described in the Methods section. Consistent with the previous study [17], treatment with STZ/HFD significantly increased fed and fasting glucose levels, diminished glucose and insulin tolerance, as well as elevated total cholesterol and triglyceride concentrations in both FGF21 KO mice and WT controls. However, these effects were further augmented in FGF21 KO mice compared with those in WT mice (Supplementary Figure S1).

On the other hand, no significant difference in body weight was observed between KO and WT mice treated with STZ/HFD, whereas the ratio of heart weight to body weight in STZ/HFD-treated FGF21 KO mice was markedly increased as compared with STZ/HFD-treated WT controls (Figure 2A). Consistent with these results, histological analysis by H&E staining also revealed that FGF21 deficiency accelerated cardiomyocyte hypertrophy in STZ/HFD-induced diabetic mice compared with WT mice under the same treatment (Figure 2B). Furthermore, FGF21 deficiency also increased the expression of atrial natriuretic peptide (ANP) and angiotensinogen (ANG) in STZ/HFD-treated mice compared with the control group (Figure 2C), suggesting that the absence of FGF21 is related to cardiac hypertrophy in STZ/HFD-induced diabetic mice.

In line with the histological change in cardiac tissues, we next explored the change in cardiac functions in STZ/HFD-induced diabetic mice. As expected, FGF21 deficiency further accelerated cardiac dysfunction in STZ/HFD-treated diabetic mice, as evidenced by increased LV end-diastolic and -systolic diameters, LV end-diastolic and -systolic volumes, and decreased ejection fraction (Table 1), compared with those in WT mice under the same treatment. On the other hand, tissue and pulse Doppler imaging analysis indicated that FGF21 deficiency decreased E-wave velocity and increased A-wave velocity as well as the E/E′ ratio in STZ/HFD-induced mice compared with WT controls (Table 2), suggesting that FGF21 deficiency accelerated diastolic dysfunction in STZ/HFD-treated mice. Taken together, these data indicated that FGF21 deficiency could increase the susceptibility for the pathogenesis of DCM in mice.

As cardiac fibrosis is one of the most important pathogenic routes of DCM, we measured cardiac collagen accumulation between FGF21 KO and WT controls. The results of Sirius Red staining indicated that FGF21 deficiency distinctly increased cardiac fibrosis and collagen accumulation in STZ/HFD-treated mice (Figure 2D). In line with these results, gene expression analysis also demonstrated that cardiac mRNA expression levels of collagen-related genes including Col-1, MHC-β, α-smooth muscle actin (α-SMA), and transforming growth factor β (TGF-β) as well as fibrosis-related genes fibronectin (FN) were markedly upregulated in STZ/HFD-treated FGF21 KO mice, compared with those in WT mice with the same administration (Figure 2E), suggesting that FGF21 deficiency accelerates cardiac fibrosis in diabetic mice.

Compelling evidence demonstrates that the development of DCM is related to the induction of cardiac oxidative stress, as characterized by the production of ROS, leading to mitochondrial dysfunction, which in turn promotes cardiac oxidative stress [29,30]. Therefore, we investigated the effect of FGF21 deletion on the production of ROS and mitochondrial dysfunction in STZ/HFD-induced diabetic mice. As expected, cardiac ROS levels detected by DHE staining (Supplementary Figure S2A,B) and the percentage of apoptotic cardiomyocytes determined by TUNEL (Supplementary Figure S2C,D) were significantly higher in STZ/HFD-treated FGF21 KO mice than those in WT controls. Furthermore, a significant attenuation in the activities of MRC complexes I and V, but not complexes II + III and IV, was observed in STZ/HFD-treated WT mice, and these effects were further augmented in STZ/HFD-treated FGF21 KO mice (Supplementary Figure S2E).

Considering that a pro-inflammatory status is often associated with the development of cardiac hypertrophy, we also investigated the effects of FGF21 absence on cardiac and systemic inflammation. Our results indicated that the mRNA levels of several inflammatory cytokines including TNF-α, IL-6, and MCP-1 were increased in STZ/HFD-treated WT mice accompanied by cardiac hypertrophy. Interestingly, these inductions were further worsened in STZ/HFD-treated FGF21 KO mice (Supplementary Figure S2F). Furthermore, a similar pattern was also observed in the serum levels of these inflammatory factors (Supplementary Figure S2G).

To further investigate the role of FGF21 in DCM, 8-week-old FGF21 KO male mice were induced to a diabetic model, and then infected with AAV encoding mouse FGF21 or GFP, respectively. As expected, AAV-mediated FGF21 overexpression significantly elevated the circulating FGF21 level (Supplementary Figure S3A), ameliorated diabetes status, as evidenced by decreased fed and fasting glucose levels, as well as improviedglucose and insulin tolerance compared with AAV-GFP controls (Supplementary Figure S3B–E). In- ine with these results, we next examined the changes in cardiac function after treatment with AAV-FGF21. Consistent with the improvement of diabetic profiles, AAV-mediated overexpression of FGF21 significantly improved systolic dysfunction in STZ/HFD-induced diabetic mice, shown as decreased LV end-diastolic and -systolic diameters, LV end-diastolic and -systolic volumes, and increased ejection fraction (Table 3). In addition, overexpression of FGF21 also reversed diastolic dysfunction in STZ/HFD-induced diabetic mice (Table 4). Taken together, these data demonstrated that FGF21 could markedly improve cardiac dysfunction in STZ/HFD-induced diabetic mice.

On the other hand, AAV-mediated overexpression of FGF21 dramatically attenuated cardiac collagen accumulation and fibrosis in STZ/HFD-induced diabetic mice as determined by H&E or Sirius Red staining respectively (Figure 3A), and markedly decreased cardiac mRNA levels of collagen- and fibrosis-related genes including Col-1, MHC-β, α-SMA, TGF-β, and FN (Figure 3B). Furthermore, treatment with AAV-FGF21 also reversed the elevated cardiac ROS content (Figure 3C,D) and cardiomyocyte apoptosis (Figure 3C,E), improved the reduction in MRC complex I and V activities (Figure 3F), and inhibited cardiac mRNA levels of inflammatory genes including TNF-α, IL-6, and MCP-1 (Figure 3G) as well as their circulating levels (Figure 3H).

Previous studies have demonstrated that β-klotho is indispensable for FGF21 bioactivity [31]. To investigate the role of β-klotho in FGF21 protection against DCM, we first examined the expression of β-klotho in cardiac tissues of STZ/HFD-induced diabetic mice and control mice. Interestingly, a significant upregulation of β-klotho expression was observed in STZ/HFD-treated WT mice compared with the controls (Supplementary Figure S4A). To investigate whether HG affects the expression of β-klotho in cardiomyocytes, primary mouse cardiomyocytes were directly exposed to HG (33 mmol/l). Surprisingly, incubation of primary cardiomyocytes with HG led to a progressive increase in β-klotho (Supplementary Figure S4B) accompanied by a time-dependent increase in intracellular ROS production (Supplementary Figure S4C).

To further explore the role of β-klotho in FGF21 protection against DCM, primary cardiomyocytes were infected with adenovirus encoding siRNA-β-klotho before treatment with HG and FGF21. As expected, treatment with rmFGF21 in primary cardiomyocytes significantly restored HG-induced elevated intracellular ROS content and mRNA levels of TGF-β, IL-6, and FN (Supplementary Figure S4D–H). However, these protective effects of FGF21 against HG-induced cytotoxicity were strongly abrogated by siRNA-mediated knockdown of β-klotho (Supplementary Figure S4D–H), suggesting that β-klotho is indispensable for FGF21 protection against HG-induced cardiomyocyte injury.

Previous studies have demonstrated that AMPK, a major cellular sensor of energy availability, is involved in the progression of cardiac injury caused by different factors [32–34]. To explore whether AMPK is involved in FGF21 against diabetes-induced cardiac injury, we first evaluated AMPK activity in FGF21 KO and WT mice. Immunoblotting analysis demonstrated that the phosphorylation level of AMPK and its downstream target ACC were significantly decreased in STZ/HFD-induced diabetic mice, compared with those in the controls. Furthermore, FGF21 deficiency further accelerated the decrease in cardiac p-AMPK and p-ACC levels in STZ/HFD-induced diabetic mice (Figure 4A,B). By contrast, treatment with AAV-FGF21 markedly reversed the reduction in cardiac p-AMPK and p-ACC levels in STZ/HFD-treated mice (Figure 4C,D), suggesting that the protective effect of FGF21 against diabetes-induced cardiac injury is related to AMPK activity in mice.

To further determine the role of AMPK in FGF21 against diabetes-induced cardiac injury, we examined the direct effect of AMPK in FGF21 protection against HG-induced cardiomyocyte injury. As expected, incubation of primary mouse cardiomyocytes with HG significantly decreased p-AMPK levels (Figure 5A), accompanied by a drastic increase in intracellular ROS accumulation and cardiomyocyte apoptosis, an obvious decrease in MRC complex I and V activities, as well as a marked elevation of mRNA expression levels of TGF-β, FN, and ANP (Figure 5B–E). Interestingly, these HG-induced effects were markedly abrogated by treatment with rmFGF21 (Figure 5B–E). However, these protective effects of FGF21 against HG-induced cardiomyocyte injury were markedly blunted by genetic inhibition of AMPK activities with infection of adenovirus-mediated DN AMPK (Figure 5B–E), suggesting that AMPK plays an indispensable role in FGF21 protection against HG-induced cardiomyocyte injury.

PON1, a member of the paraoxonase subfamily, is involved in the onset and development of cardiovascular diseases [35–37]. To explore whether PON1 participates in FGF21 protection against DCM, we first tested the expression of PON1 in both STZ/HFD-treated FGF21 KO and WT mice. As expected, the cardiac PON1 protein and its mRNA levels, as well as arylesterase activity and PON1 activity, were significantly decreased in STZ/HFD-treated WT, and these effects were further down-regulated in STZ/HFD-treated FGF21 KO mice (Figure 6A–D). Intriguingly, treatment with AAV-FGF21 markedly reversed the reduction in cardiac PON1 expression as well as its arylesterase and paraoxonase activities in STZ/HFD-treated mice (Figure 6E–H), suggesting that PON1 may play a pivotal role in FGF21 protection against DCM.

To further determine the role of PON1 in FGF21 against DCM, we next tested the direct effect of PON1 in FGF21 protection against HG-induced cardiomyocyte injury. In line with our previous observations in mice, incubation of primary mouse cardiomyocytes with FGF21 significantly enhanced PON1 expression in a dosage- and time-dependent manner (Figure 7A,B). However, treatment of primary cardiomyocytes with FGF21 reversed HG-elevated intracellular ROS accumulation, decreased MRC complex I and V activities, upregulated mRNA levels of TGF-β, FN, and IL-6, and these FGF21-induced effects were strongly blunted by genetic inhibition of PON1 with siRNA (Figure 7C–F), suggesting that PON1 may at least, in part, mediate the protective effect of FGF21 against HG-induced cardiomyocyte injury.

Based on our results that FGF21 alleviated HG-induced oxidative stress and fibrosis by activating the AMPK signaling pathway, we further explored whether AMPK is involved in the regulation of FGF21-induced PON1 expression in cardiomyocytes. As expected, FGF21-induced PON1 expression was strongly blunted by infection with adenovirus-mediated expression of DN AMPK (Figure 8A), which was accompanied by an increased intracellular ROS accumulation, upregulated the expression of TGF-β, FN, and IL-6, as well as inhibited MRC complex I and V activities (Figure 8B–E). Interestingly, these effects were strongly reversed when infected with adenovirus-PON1 (Figure 8B–E). Taken together, these data demonstrate that FGF21-induced PON1 expression is mediated by the AMPK signal pathway, and PON1 mediates the protective effect of FGF21 against HG-induced cytotoxicity in cardiomyocytes independently.

Diabetes is currently a serious global issue. Amongst all the complications of diabetes, DCM is considered to be the primary cause of mortality in diabetic patients. A recent study involving STZ-induced type 1 diabetic mice indicated that FGF21 deficiency rendered mice more susceptible to develop DCM [15]. However, the exact mechanism of FGF21 protection against DCM is still obscure. The present study demonstrates that FGF21 deficiency lessens AMPK activation and PON1 expression and promotes cardiac injury in STZ/HFD-induced diabetic mice. Overexpression of FGF21 significantly increases AMPK activation and PON1 content in mice, accompanied by improvement of cardiac injury. Furthermore, treatment with FGF21 reverses HG-induced oxidative stress, mitochondrial dysfunction, and inflammatory response by activating the AMPK-PON1 axis in primary cardiomyocytes. Taken together, these data demonstrate that the FGF21-AMPK-PON1 axis couples the protective effects of FGF21 in the protection of DCM in mice.

FGF21, a member of the endocrine FGF subfamily, is a metabolic hormone with pleiotropic effects on glucose and lipid metabolism and insulin sensitivity. Animal-based studies demonstrate that FGF21 lowers glucose and triglyceride levels, and exhibits insulin-sensitizing properties in diabetic rodents and monkeys [6,38]. A recent study indicated that loss of FGF21 worsened DCM in STZ-induced type 1 diabetic mice [15]. In line with this observation, we also found that FGF21 deficiency accelerated cardiomyopathy accompanied by ameliorated cardiac hypertrophy (Figure 2), elevated cardiac oxidative stress, improved mitochondrial dysfunction (Figure 3), and enhanced inflammation and fibrosis (Figure 4) in STZ/HFD-induced diabetic mice. On the other hand, we also found that overexpression of FGF21 significantly attenuates DCM, followed by decreased cardiac oxidative stress, enhanced MRC complex activities, and inhibited inflammation and fibrosis in STZ/HFD-induced diabetic mice (Figure 5). Taken together, these data indicate that FGF21 protection against DCM may be related to improved mitochondrial function. To support this, FGF21 has been reported to regulate mitochondrial function by activating the AMPK-Sirt1-PGC1α pathway [39].

Klotho, a transmembrane protein, provides some control over the sensitivity of an organism to insulin and appears to be involved in ageing [40]. Recently, β-klotho, a member of the klotho family, was found to physically interact with FGF receptors 1c and 4, thereby increasing the ability of these FGF receptors to bind with FGF21 and activate the downstream signaling pathway [41]. Knockdown of β-klotho by siRNA in adipocytes diminishes glucose uptake induced by FGF21 [41]. These findings indicate that β-klotho plays an indispensable role in the FGF21-mediated maintenance of glucose hemostasis and pathogenesis of diabetes. In the present study, different from a previous report that β-klotho expression in pancreatic islets was significantly attenuated when directly treated with HG [42], cardiac β-klotho expression was significantly upregulated in STZ/HFD-treated WT mice, and this elevated profile was markedly abrogated in FGF21-deficient mice under the same status, suggesting that FGF21 deficiency may affect cardiac expression of β-klotho in mice. Furthermore, our in vitro data also indicated that genetic inhibition of β-klotho by RNA interference almost reversed the protective effects of FGF21 against HG-induced oxidative stress and mitochondrial dysfunction, suggesting that β-klotho is essential for FGF21 protection against diabetes-induced cardiac injury.

Mounting evidence indicates that DCM is closely associated with increased oxidative stress and mitochondrial dysfunction. Under physiological conditions, ROS production by cardiomyocytes is regulated by a number of enzymes and antioxidants, and there is a physiological balance between ROS and antioxidants. However, under pathological conditions, such as diabetes, this balance is disrupted by hyperglycemia followed by overproduction of ROS, and then initiates harmful effects on cardiomyocytes, resulting in mitochondrial dysfunction, myocardial cell death, subsequent hypertrophy and fibrosis, and eventually leads to cardiac dysfunction [3]. PON1 was first discovered through its ability to hydrolyze and therefore detoxify organophosphorus compounds, which are widely used as pesticides and nerve gases. Due to its ability to destroy oxidized lipids, PON1 appears to play a role in some certain diseases. Recently, a number of studies have indicated that PON1 presents multiple biofunctions including anti-inflammatory, antioxidative, anti-atherogenic, antidiabetic, antimicrobial, and organophosphate-hydrolyzing properties. Therefore, it was considered as a potential agent against cardiovascular diseases due to these biocharacteristics [43]. In the present study, we first found that PON1 is involved in the pathogenesis of DCM. Cardiac PON1 is significantly decreased in STZ/HFD-induced WT diabetic mice followed by an increase in cardiac ROS production and a decrease in MRC complex activities, and the magnitude of decrease in PON1 content is further augmented in STZ/HFD-induced FGF21-deficient mice. In contrast, overexpression of FGF21 dramatically increases cardiac PON1 expression accompanied by a reduction in ROS level and inhibition of decreased MRC complex activities. Our in vitro data also indicate that the protective effects of FGF21 against HG-induced cytotoxicity are also strongly abrogated by inhibition of PON1 in primary mouse cardiomyocytes, suggesting that the protective effects of FGF21 against diabetes-induced cardiac damage are at least in part mediated by the antioxidative effect of PON1.

AMPK is a serine-threonine kinase that acts as an energy sensor in various cellular types. AMPK acts as a metabolic master switch regulating several intracellular systems including the cellular uptake of glucose, the β-oxidation of fatty acids, and the biogenesis of glucose transporter 4 and mitochondria [44,45]. The energy-sensing capability of AMPK can be attributed to its ability to detect and react to fluctuations in the AMP:ATP ratio that take place during rest and exercise [45,46]. AMPK signaling pathways are activated by adipokines, including adiponectin and leptin secreted from adipose tissues, which play several distinct but important physiological and pathological roles in the body [47,48]. AMPK is significantly activated in response to cardiac energy imbalance during myocardial ischemia and myocardial infarction, and inactivation of AMPK was found to enhance myocardial injury during ischemia–reperfusion in mice [32,49]. Recently, we indicated that FGF21 protects against cardiac apoptosis in a type 1 diabetic mouse model by activating the extracellular signal-regulated kinase (ERK) 1/2-p38 mitogen-activated protein kinase (MAPK)-AMPK pathway [19], and pharmaceutical inhibition of AMPK led to a significant decrease in FGF21-induced cardioprotection and restoration of cardiac function in response to global ischemia [12]. In the present study, our in vivo data indicate that cardiac activation of AMPK was markedly upregulated in STZ/HFD-induced diabetic mice. However, this elevation of AMPK activation was strongly attenuated in FGF21 KO mice, suggesting that FGF21 may be involved in the regulation of AMPK activity in response to restoring cardiac-energy homeostasis in STZ/HFD-induced diabetic mice. On the other hand, FGF21 inhibits palmitate-induced down-regulation of AMPK activation in cultured cardiomyocytes [19]. Consistent with these reports, our in vitro results also demonstrated that treatment with FGF21 significantly improved the activation of AMPK in response to HG-induced cytotoxicity in primary cardiomyocytes, and genetic inhibition of AMPK signaling markedly abrogated the protective effect of FGF21, suggesting that AMPK plays an indispensable role in FGF21 protection against HG-induced cardiomyocyte injury.

In summary, our present study demonstrates that FGF21 protection against DCM is mediated by activation of the AMPK signaling pathway and enhanced PON1 expression, which, in turn, inhibits HG-induced oxidative stress and leads to alleviation of local inflammation, fibrosis, and cardiomyocyte apoptosis, thereby protecting against DCM (Supplemental Figure S5). Further studies on large humanoid animals and clinical investigations are warranted to validate these findings in rodent models.

• FGF21 is a metabolic hormone with pleiotropic effects on glucose metabolism and insulin sensitivity.

• In the present study, our data indicate that FGF21 protecting against DCM is mediated at least in part by activating AMPK-PON1 axis.

• Our findings enrich our knowledge on FGF21 and metabolic regulation, and reignite hope for the future development of effective antidiabetic complication therapies.

We thank Dr Ying Zhi and Dr Dewei Ye for their technical support related to the examination of heart function and the construction of the AAV-FGF21 virus system.

This work was supported by the National Natural Science Foundation of China General Program [grant number 81471075]; and the National Natural Science Foundation of China Major Program [grant number 91439123].

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

F.W., B.W., S.Z., L.S., and X.P. researched the data. L.S., R.X., and Y.W. wrote the manuscript and researched the data. Y.W. and F.G. reviewed/edited the figures. X.L. and Z.L. contributed to the discussion and reviewed/edited the manuscript.

AAV

adeno-associated virus

ACC

acetyl-CoA carboxylase

AMPK

AMP-activated protein kinase

ANG

angiotensinogen

ANP

atrial natriuretic peptide

Col-1

collagen-1

DCM

diabetic cardiomyopathy

DHE

dihydroethidium

DN

dominant-negative

ECHO

echocardiography

FGF21

fibroblast growth factor 21

FN

fibronectin

HFD

high-fat diet

HG

high glucose

H&E

hematoxylin-eosin

IL-6

interleukin-6

MCP-1

monocyte chemoattractant protein 1

MRC

mitochondrial respiratory chain

PON1

paraoxonase 1

rmFGF21

recombinant mouse fibroblast growth factor 21

ROS

reactive oxygen species

STZ

streptozotocin

TGF-β

transforming growth factor β

TNF-α

tumor necrosis factor α

α-SMA

α-smooth muscle actin