Liver X receptor agonist treatment attenuates cardiac dysfunction in type 2 diabetic db/db mice

Background Liver X receptor (LXR) plays a critical regulatory role in metabolism and inflammation, and has been demonstrated to be involved in cardiovascular physiology/pathology. In the present study, we investigated the effect of GW3965, a potent LXR agonist, on diabetic cardiomyopathy (DCM) in type 2 diabetic db/db mice. Methods and Results Non-diabetic db/+ mice and diabetic db/db mice received either vehicle or LXR agonist GW3965 for 12 weeks. Systemic insulin resistance was evaluated by glucose tolerance test and homeostasis model assessment for insulin resistance. Endpoint cardiac function was assessed by echocardiography and catheterization. Ventricular tissue was collected for histology and gene/protein expression analysis. Untreated db/db diabetic mice exhibited diastolic dysfunction with adverse structural remodeling (including myocardial fibrosis and increased apoptosis). Treatment with GW3965 remarkably attenuated myocardial dysfunction and structural remodeling in diabetic db/db mice. Mechanistically, GW3965 restored Akt phosphorylation and inhibited MAP kinases phosphorylation, and reduced oxidative/nitrative stress and inflammation response in the diabetic myocardium. Conclusions Our data demonstrate that GW3965 exerts a cardioprotective effect against DCM by (at least in part) attenuating insulin resistance, modulating Akt and MAP kinases pathways, and reducing oxidative/nitrative stress and inflammatory response. These findings strongly suggest that LXR agonist may have therapeutic potential in treating DCM.


Background
The incidence and prevalence of diabetes mellitus are growing rapidly in societies around the world. Type 2 diabetes mellitus (T2DM) accounts for 90-95% of all diagnosed diabetes in adults [1]. Growing evidence has shown that diabetes mellitus, independent from other risk factors such as coronary artery disease and hypertension, can affect cardiac structure and function, which supports the existence of diabetic cardiomyopathy (DCM) [2]. As an independent diabetic cardiac complication, DCM is defined as diabetes-caused pathologic abnormalities including myocardial metabolic disturbance, oxidative/nitrative stress, inflammation, cardiomyocyte apoptosis, left ventricular dysfunction and structural remodeling [3]. Although treatment for DCM including improving glycemic control and restoring cardiovascular function is currently available to diabetic patients in clinical practice, therapeutic outcomes are far from satisfactory and the incidence of diabetes-induced cardiac dysfunction continues to escalate [3,4]. Thus, there is a great medical need to develop novel pharmacological or molecular interventions to treat left ventricular dysfunction and remodeling in DCM.
Liver X receptors (LXRs), including two different but highly homologous LXR isoforms (LXRα and LXRβ), are ligand-activated transcriptional factors belonging to the nuclear receptor superfamily [5]. LXRα is highly expressed in metabolically active tissues, such as liver, kidney, adipose, and intestines. LXRβ is ubiquitously expressed throughout the body [5]. Recently, by regulating metabolic and inflammatory pathways, LXR has been considered as a potential pharmacological target in the pathogenesis of cardiovascular and metabolic diseases [6]. Two synthetic LXR agonists, GW3965 and T0901317, have been reported to prevent atherosclerosis, inhibit inflammation, attenuate myocardial hypertrophy, and reduce ischemia/reperfusion injury [7][8][9][10][11]. Moreover, activation of LXR by T0901317 mitigates high glucose-induced oxidative stress, and apoptosis in cardiomyocytes in vitro [12]. However, the potential of LXR activation to attenuate the structural and functional defects caused by DCM in vivo have not been investigated. Therefore, the aims of the current study were to 1) investigate whether the LXR agonist GW3965 can protect the diabetic heart against adverse changes using the db/db mouse model of T2DM; and 2) clarify the downstream signaling mediating its effect in DCM.

Animals and treatment
Experimental protocols complied with the National Institutes of Health Guidelines on the Use of Laboratory Animals, and were approved by the Institute's Animal Ethics Committee. Male diabetic (db/db) mice and their nondiabetic littermates (db/+) were obtained from the SLAC Experimental Animal Center (Shanghai, China) and were housed at 22 ± 1°C, adherent to a 12 hour light-dark cycle. All the animals were provided with food and water ad libitum. At 8 weeks of age, the db/+ and db/db mice were randomized into four groups: (1) control db/+ mice (db/+); (2) db/+ mice + GW3965 (db/+ GW); (3) db/db mice (db/db); (4) db/db mice + GW3965 (db/db GW). The mice were treated with GW3965 (20 mg/kg intraperitoneally) or vehicle daily for 12 weeks prior to sacrifice and tissue collection. GW3965 at this dose, which was chosen based upon our pilot study data and the published literature [11,13,14], GW3965 at this dose effectively invokes LXR activity without inducing observable hemodynamic changes in animal studies [11,13,14]. After 12 weeks, animals were subjected to hemodynamic measurements (described below), and hearts were excised and snap-frozen in liquid nitrogen for biochemical determinations, or fixed in formalin for histological evaluations. At the endpoint, mice were fasted overnight and serum samples were collected. Plasma glucose, total cholesterol (TC), and serum triglyceride (TG) were determined by an auto-biochemical analysis system (Chemix-180, Sysmex, Japan).

Glucose Tolerance Test (GTT) and Homeostasis Model Assessment for Insulin Resistance (HOMA-IR)
Briefly, mice were fasted overnight (14-16 h). GTT was performed as described previously [16,17]. Glucose solution was administered via an intraperitoneal injection at a dose of 2 g/kg body weight, and the blood glucose level was measured from tail snipping at 0, 30, 60, 90, and 120 min after the initial glucose loading. Blood glucose level was determined using a One-Touch Profile portable blood glucose monitor (Roche, Mannheim, Germany). The area under the curve of the glucose concentrations (AUCg) was calculated. Serum insulin was measured by ELISA (Millipore, Billerica, MA) [18]. HOMA-IR was calculated using the following formula: HOMA-IR (mmol/L × μU/mL) = fasting glucose (mmol/L) × fasting insulin (μU/mL) /22.5 [18].

In situ detection of apoptosis in heart tissue
Myocardial apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) technique via an In Situ Cell Death Detection Kit (Roche Diagnostics) as described previously [19,20]. Results were expressed as the percentage of apoptotic cells among the total cell population.

Detection of caspase-3 activity in heart tissue
Cardiac caspase-3 activity was measured via caspase-3 Colorimetric Assay Kit (Millipore, Billerica, MA) as previously described [15,21,22]. Briefly, 100 μg of total protein from tissues was loaded and incubated with 25 μg Ac-DEVD-pNA as a colorimetric-specific substrate at 37°C for 1.5 hours. Then, pNA cleaved from DEVD by caspase-3 was quantified by a microplate reader (BioTek, Winooski, VT) at 405 nm. Changes of caspase-3 activity in db/db tissue samples were calculated and compared with the mean value from control db/+ mice tissue. Data were expressed as nmol pNA /h /mg protein.

Hemodynamic measurements
Left ventricular hemodynamics was evaluated after 12 weeks by catheterization as previously described [23]. In brief, a micromanometer-tipped catheter (1.4 F, SPR 835; Millar Instruments, Houston, TX) was inserted through the right carotid artery into the aorta of an anaesthetized mouse and carefully introduced into the left ventricle (LV). The transducer was connected to a Power Laboratory system (AD Instruments, Castle Hill, New South Wales, Australia) and variables derived from catheterization included maximal ascending and descending rates of left ventricular pressure (±dP/dt), left ventricular end-systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), and heart rate.

Echocardiographic measurements
At 20 weeks of age, mice were anaesthetized with 1.5% isoflurane, and two-dimensional echocardiographic views of the mid-ventricular short axis were obtained at the level of the papillary muscle tips below the mitral valve (Vevo 770, VisualSonic, Toronto, Canada). Variables measured on Mmode echocardiography including LV wall thickness, LV chamber dimensions, and systolic and diastolic function were analyzed as described previously [24].

Sirius red staining
LV myocardial sections were stained with 0.1% picric sirius red (Sigma-Aldrich, St Louis, MO) for fibrosis detection, as previously described [25]. The severity of cardiac fibrosis was evaluated after sirius red staining at 20 × magnification with the use of Image-pro plus 6.1 software (Media Cybernetics, Bethesda, MD). Collagen-positive area was normalized to the total cross-sectional area of left ventricle and was expressed as a percentage. Areas containing blood vessels and perivascular interstitial cells were excluded from fibrosis quantification [26].

Measurement of oxidative stress generation in heart tissue
Myocardial reactive oxygen species (ROS) generation was measured by confocal microscope via in situ DHE stain or lucigenin-enhanced chemiluminescence. For DHE stain, unfixed frozen cross-sections (5 μm) was incubated with DHE (5 μmol/L) at 37°C for 30 minutes in a humidified chamber protected from light, followed by 5 minutes of PBS washing to remove non-intercalated ethidium bromide molecules. Images were obtained and analyzed via Leica laser scanning confocal microscope (Leica TCS SP5 II). NADPH oxidase activity within the heart homogenates was measured by lucigenin-enhanced chemiluminescence via luminometer as previously described [11,21,22]. The lucigenin concentration in the final reaction mixture was 0.25 mmol/L, and NADPH-dependent superoxide production was expressed as relative light units (RLU) per mg per second (RLU • mg -1 • s -1 ).

Assessment of inflammatory cytokines in cardiac tissue
Tumor necrosis factor alpha (TNF-α) and interleukin 1beta (IL-1β) were quantified using an ELISA kit (Invitrogen, Camarillo, CA) per manufacturer's instructions. The tissue supernatant fluids were added to each well, and treated with detection antibody, supplemented with substrate and stop solution. TNF-α and IL-1β levels were determined by microplate reader (450 nm).

Statistical analysis
All values in the text and figures are presented as the mean ± SEM of independent experiments from given nsizes. Statistical significance of multiple treatments was determined by one-way analysis of variance (ANOVA) followed by Tukey's post hoc analysis using GraphPad Prism 5 software (San Diego, California). Probabilities of 0.05 or less were considered to be statistically significant (2-tailed).

Results
Both LXRα and LXRβ are expressed in adult heart tissue, but LXRα is selectively upregulated by hyperglycemia in db/db mice Both LXRα and LXRβ subtypes were detected in cardiac tissue, as demonstrated by both Western blot (Figure 1a) and real-time PCR (Figure 1b). Interestingly, endogenous LXRα protein level significantly increased in the db/db and db/db GW group, whereas LXRβ expression remained mostly unaffected.
LXR agonist GW3965 attenuates the phenotype of type 2 diabetes in db/db mice We first investigated whether the activation of LXR could suppress hyperglycemia and other metabolic abnormalities in diabetic mice. As illustrated in Table 1, the db/db group weighed more than the db/+ group, and demonstrated derangements of serum metabolic markers such as glucose, TC and TG. Chronic treatment of 8-week-old male diabetic db/db mice with LXR agonist GW3965 lowered body weight, blood glucose levels, and plasma TC levels. However, GW3965 showed no influence on the increased plasma TG level in diabetic mice. These findings indicate that LXR activation by GW3965 improved the metabolic disorders in this mouse model of T2DM.
LXR agonist GW3965 improves glucose tolerance and insulin sensitivity in db/db mice We next tested the effect of LXR agonist on insulin resistance in db/db diabetic mice. At 20 weeks of age, there were no statistically significant differences in the GTT, the AUCg and HOMA-IR index between the db/+ and db/+ GW group (Figure 2). However, the levels of GTT, AUCg, and HOMA-IR index were markedly deteriorated in db/db diabetic mice, which were significantly reversed by the administration of GW3965 for 12 weeks (Figure 2).

LXR agonist GW3965 inhibits myocardial apoptosis and cardiac dysfunction in db/db mice
In an attempt to determine the cardioprotective role of LXR agonist, we investigated the effects of GW3965 on cellular apoptosis and cardiac function in type 2 diabetic db/db mice. Our results demonstrated that compared with the db/+ group, db/db mice clearly showed more TUNEL-positive particles (Figure 3, a and b), increased cleaved caspase-3 expression, enhanced caspase-3 activity (Figure 3, c and d) in cardiomyocytes, and deteriorated left ventricular dysfunction (Figure 4). GW3965 treatment significantly decreased cardiomyocyte apoptosis and improved cardiac function (Figures 3 and 4).

LXR agonist GW3965 reduces cardiac fibrosis in db/db mice
By sirius red staining, we found that db/db mice exhibited more severe cardiac fibrosis than the control group ( Figure 5, a and b). This induction of fibrosis was significantly ameliorated after GW3965 treatment for 12 weeks ( Figure 5, a and b). Moreover, real-time PCR analysis revealed significant increases in the expression of profibrotic genes (TGF-β1 and collagen-1A1) in diabetic hearts, which were attenuated by GW3965 ( Figure 5, c and d).
LXR agonist GW3965 attenuates myocardial oxidative stress and nitrative stress in db/db mice To further determine the underlying mechanisms of LXR agonist's protective action, we investigated the effects of GW3965 on oxidative/nitrative stress in the diabetic myocardium. GW3965 significantly attenuated ROS production in db/db mice (Figure 6, a and b) and inhibited the expression of the NADPH oxidase subunit gp 91phox (Figure 6c). Moreover, GW3965 significantly reduced tissue nitrotyrosine content (a well-accepted footprint of in vivo nitrative stress, Figure 6, d and e), and inhibited iNOS expression (Figure 6f ). Collectively, these results demonstrate that GW3965 attenuated diabetes-induced oxidative/nitrative stress.

LXR agonist GW3965 suppresses diabetes-induced myocardial nuclear factor-κB activation and inflammation
To investigate whether LXR agonist provided cardioprotection by inhibiting the inflammatory response in the diabetic myocardium, we investigated the effect of GW3965 on inflammatory cytokine production. Protein levels of NF-κB p65 were markedly increased in the myocardium of db/db mice (Figure 7a). The expression of proinflammatory cytokines, including TNF-α and IL-1β, was significantly augmented in db/db mice (Figure 7, b and c). Furthermore, treatment with GW3965 significantly decreased nuclear NF-κB p65 expression and proinflammatory cytokine (TNF-α and IL-1β) levels in db/db mice (Figure 7). These results indicate that the anti-inflammatory effects of LXR agonist contribute to cardioprotection against DCM.

LXR agonist GW3965 ameliorates the impairment of insulin/ Akt signaling pathway and mitigates diabetes-induced activation of MAP kinases
To further investigate the cellular mechanisms by which LXR activation may attenuate diabetes-induced insulin resistance and cardiac oxidative stress, we evaluated insulin/ Akt signaling and MAPK pathways, which are the most important pathways involved in insulin resistance and oxidative/nitrative stress. Serine phosphorylation of Akt (serine 473) was impaired in the myocardium of db/db mice. However, 12-week treatment with GW3965 significantly restored Akt activation in db/db mice (Figure 8a). Likewise in diabetic myocardium, an increase in the activation of p38 MAPK (Figure 8b) and JNK (Figure 8c) could be observed. Treatment with GW3965 significantly inhibited diabetesinduced p38 MAPK and JNK phosphorylation without  altering their protein levels (Figure 8, b and c). These data suggest that LXR activation alleviated DCM mainly by modulating Akt and MAP kinases pathways in type 2 diabetes.

Discussion
The major findings emanating from the current study are as follows: (1) Both LXRα and LXRβ subtypes were detected in adult cardiac tissue, but LXRα was selectively upregulated by hyperglycemia; (2) GW3965 ameliorated metabolism and improved glucose tolerance and insulin sensitivity; (3) GW3965 protected the heart in the db/db mouse model of type 2 diabetes from the development of diastolic dysfunction, cell death, and cardiac fibrosis; and (4) GW3965 also restored Akt phosphorylation and inhibited MAP kinases phosphorylation, and reduced oxidative/ nitrative stress and inflammation response in the diabetic myocardium. Taken together, these results suggest that LXR agonist GW3965 may have great therapeutic potential in the treatment of DCM.
Originally cloned from a rat liver cDNA library and identified as an orphan nuclear hormone receptor [27], LXR is highly expressed in the enterohepatic tissue, playing a pivotal role in cholesterol and lipid metabolism, glucose homeostasis, and inflammatory response [5]. Recent evidence suggests the presence of LXR in the cardiovascular system and its significant role in cardiovascular physiology/ pathology [8][9][10][11]. In the myocardium, the expression of LXRα was increased in streptozotocin-induced diabetic rats [28]. LXR agonist T0901317 attenuated high glucoseinduced cardiomyocyte apoptosis in vitro [12]. However, the role of LXR in DCM in vivo remains unknown. To the best of our knowledge, the current study provides the first direct evidence demonstrating that GW3965 protects cardiomyocytes against hyperglycemia-induced chronic adverse changes in vivo. These data support the notion that LXR agonist may serve as a novel therapeutic modality in the management of DCM.
Hyperglycemia and insulin resistance have long been considered a central component in the pathogenesis of DCM [29,30]. In insulin resistance states, impaired myocardial GTT was performed at baseline and 12 weeks after treatment with GW3965 (n =7-9). c-d. Area under the curve of glucose levels during the GTT were also calculated (n =7-9 per group). e. HOMA-IR index was detected (n =7-9). **P < 0.01 vs db/+ group; ## P < 0.01 vs db/db group. Abbreviations: GTT, glucose tolerance test; GW, GW3965; HOMA-IR, homeostasis model assessment for insulin resistance.
glucose uptake has been shown to provoke myocardial dysfunction [31]. In addition, insulin resistance independently predicts mortality in diabetic patients with heart failure [3]. Multiple lines of evidence have indicated that LXR is an important regulator of glucose metabolism in different animal models of T2DM. Previous studies showed that pharmacologic LXR activation by GW3965 improved glucose tolerance by limiting hepatic glucose output and improving peripheral glucose uptake in a murine model of diet-induced obesity and insulin resistance [32]. Moreover, LXR agonist GW3965 reduced blood glucose concentrations and improved insulin sensitivity in ob/ob mice [33].   Pooled data on quantification of collagen area per visual field (n =8-9). c-d. TGF-β1 and collagen 1A1 gene expression was determined by real-time PCR. Results were normalized against GAPDH and converted to fold induction relative to db/+ mice (n =6). *P < 0.05 or **P < 0.01 vs db/+ group; # P < 0.05 or ## P < 0.01 vs db/db group. Abbreviations: GW, GW3965; LV, left ventricle; TGF-β1, transforming growth factor beta1; collagen 1A1, collagen, type I, alpha 1. Figure 6 GW3965 attenuated diabetes-induced myocardial oxidative/nitrative stress. a-c. GW3965 attenuated oxidative stress in the myocardial tissues. a. Myocardial oxidative stress was measured by confocal microscopy with in situ dihydroethidium stain (n =5-6, scale bar 25 μm). b. NADPH oxidase activity was determined by lucigenin-enhanced chemiluminescence (n =6-11). c. NADPH oxidase gp 91phox gene expression was determined by real-time PCR (n =6). Results were normalized against GAPDH and converted to fold induction relative to db/+ mice. d-f. GW3965 attenuated nitrative stress in the myocardial tissues. d. Myocardial nitrative stress was assessed via nitrotyrosine levels determined by immunohistochemistry (n =5-6, scale bar 25 μm). e. Myocardial nitrotyrosine content was determined by ELISA analysis (n =6-11). f. iNOS gene expression was determined by real-time PCR (n =6). Results were normalized against GAPDH and converted to fold induction relative to db/+ mice. *P < 0.05 or **P < 0.01 vs db/+ group; # P < 0.05 or ## P < 0.01 vs db/db group. Abbreviations: GW, GW3965; RLU, relative light units; iNOS, inducible nitric oxide synthase.
The results of the present study further demonstrated that the LXR agonist GW3965 ameliorates systemic insulin resistance and myocardial dysfunction in a db/db murine model.
Hyperglycemia and insulin resistance contribute to the generation of excessive ROS/RNS which have damaging effects on myocardial function. Hyperglycemia enhances glucose oxidation and mitochondrial generation of ROS, which has been implicated as a key stimulator of these cardiac impairments [24,34,35]. Meanwhile, NO is overproduced by activated iNOS, reacts with ROS, and turns into the highly reactive ONOO -(RNS). ROS/RNS trigger myocardial apoptosis, and damage the mitochondrial membrane, leading to a "ROS-induced ROS release" vicious cycle, which further worsens myocardial damage and dysfunction [3,36]. Because of these roles of ROS/ Figure 7 GW3965 suppressed diabetes-induced inflammatory response in the myocardial tissues. a. Expression of nuclear NF-κB p65 subunit in the myocardial tissue was determined by Western blot (n =5-6). Histone level served as loading control for nuclear protein expression. Results were normalized against histone and converted to fold induction relative to db/+ mice. b-c. TNF-α and IL-1β content in the myocardial tissue was determined by ELISA analysis (n =6-8). **P < 0.01 vs db/+ group; # P < 0.05 or ## P < 0.01 vs db/db group. Abbreviations: GW, GW3965; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β. RNS, reducing oxidative/nitrative stress should be favored in the management of DCM. In this report, we demonstrate that long-term administration of GW3965 significantly decreased the expression of myocardial NADPH oxidase, as well as superoxide production and tissue nitrotyrosine content (the footprint of in vivo nitrative stress). Furthermore, we showed that GW3965 treatment inhibited the downstream inflammatory response (i.e., activation of nuclear NF-κB and proinflammatory cytokines) in diabetic myocardium in vivo. Thus, inhibiting ROS/RNS and inflammatory pathways could be an important mechanism responsible for LXR agonist-mediated cardioprotection against DCM.
There is accumulating evidence that in the setting of type 2 diabetes, insulin resistance and ROS/RNS may be coconspirators in cardiac dysfunction, each capable of triggering or worsening the other [37]. The insulin-Akt signaling and MAPK are the most important pathways involved in insulin resistance and oxidative stress [38]. It has been demonstrated the synthetic LXR agonists ameliorated insulin resistance by restoring the insulin-Akt signalling cascade and preventing JNK activation in adipocytes [39,40]. In the current study, the insulin-Akt pathway was blunted, compared with the activated MAPK pathway in the myocardium of db/db mice. Treatment with GW3965 restored Akt activation and inhibited MAP kinases phosphorylation in the diabetic myocardium, suggesting that differential regulation of Akt and MAP kinases activation are likely responsible for the aforementioned cardioprotective effects of the LXR agonist against DCM; further studies are warranted to define in more detail the complex mechanisms involved in regulation of oxidative stress, inflammatory response, and the cardioprotective effect of GW3965.

Conclusion
Our data demonstrate that the LXR agonist GW3965 exerts a protective effect on DCM by (at least in part) attenuating insulin resistance, modulating Akt and MAP kinases pathways, and reducing oxidative/nitrative stress and inflammatory response ( Figure 9). LXR, therefore, is a potentially attractive molecular target for the treatment of DCM.