Oridonin attenuates atherosclerosis by inhibiting foam macrophage formation and inflammation through FABP4/PPARγ signalling

Both lipid accumulation and inflammatory response in lesion macrophages fuel the progression of atherosclerosis, leading to high mortality of cardiovascular disease. A therapeutic strategy concurrently targeting these two risk factors is promising, but still scarce. Oridonin, the bioactive medicinal compound, is known to protect against inflammatory response and lipid dysfunction. However, its effect on atherosclerosis and the underlying molecular mechanism remain elusive. Here, we showed that oridonin attenuated atherosclerosis in hyperlipidemic ApoE knockout mice. Meanwhile, we confirmed the protective effect of oridonin on the oxidized low-density lipoprotein (oxLDL)-induced foam macrophage formation, resulting from increased cholesterol efflux, as well as reduced inflammatory response. Mechanistically, the network pharmacology prediction and further experiments revealed that oridonin dramatically facilitated the expression of peroxisome proliferator-activated receptor gamma (PPARγ), thereby regulating liver X receptor-alpha (LXRα)-induced ATP-binding cassette transporter A1 (ABCA1) expression and nuclear factor NF-kappa-B (NF-κB) translocation. Antagonist of PPARγ reversed the cholesterol accumulation and inflammatory response mediated by oridonin. Besides, RNA sequencing analysis revealed that fatty acid binding protein 4 (FABP4) was altered responding to lipid modulation effect of oridonin. Overexpression of FABP4 inhibited PPARγ activation and blunted the benefit effect of oridonin on foam macrophages. Taken together, oridonin might have potential to protect against atherosclerosis by modulating the formation and inflammatory response in foam macrophages through FABP4/PPARγ signalling.     

Long noncoding RNA THRIL promotes foam cell formation and inflammation in macrophages

Tumor necrosis factor-α (TNF-α) and heterogenous nuclear ribonucleoprotein L (hnRNPL)-related immunoregulatory lincRNA (THRIL) is a long noncoding RNA (lncRNA) involved in various inflammatory diseases. However, its role in atherosclerosis is not known. In this study, we aimed to investigate the function of THRIL in mediating macrophage inflammation and foam cell formation. The expression of THRIL was quantified in THP-1 macrophages after treatment with oxidized low-density lipoprotein (oxLDL). The effect of THRIL overexpression and knockdown on oxLDL-induced inflammatory responses and lipid accumulation was determined. THRIL-associated protein partners were identified by RNA pull-down and RNA immunoprecipitation assays. We show that THRIL is upregulated in macrophages after oxLDL treatment. Knockdown of THRIL blocks oxLDL-induced expression of interleukin-1β (IL-1β), IL-6, and TNF-α and lipid accumulation. Conversely, ectopic expression of THRIL enhances inflammatory gene expression and lipid deposition in oxLDL-treated macrophages. Moreover, THRIL depletion increases cholesterol efflux from macrophages and the expression of ATP-binding cassette transporter (ABC) A1 and ABCG1. FOXO1 is identified as a protein partner of THRIL and promotes macrophage inflammation and lipid accumulation. Furthermore, overexpression of FOXO1 restores lipid accumulation and inflammatory cytokine production in THRIL-depleted macrophages. In conclusion, our data suggest a model where THRIL interacts with FOXO1 to promote macrophage inflammation and foam cell formation. THRIL may represent a therapeutic target for atherosclerosis.     

Fatty Acid binding protein 4 is associated with carotid atherosclerosis and outcome in patients with acute ischemic stroke

Background and Purpose

Fatty acid binding protein 4 (FABP4) has been shown to play an important role in macrophage cholesterol trafficking and associated inflammation. To further elucidate the role of FABP4 in atherogenesis in humans, we examined the regulation of FABP4 in carotid atherosclerosis and ischemic stroke.

Methods

We examined plasma FABP4 levels in asymptomatic (n = 28) and symptomatic (n = 31) patients with carotid atherosclerosis, as well as in 202 subjects with acute ischemic stroke. In a subgroup of patients we also analysed the expression of FABP4 within the atherosclerotic lesion. In addition, we investigated the ability of different stimuli with relevance to atherosclerosis to regulate FABP4 expression in monocytes/macrophages.

Results

FABP4 levels were higher in patients with carotid atherosclerosis, both systemically and within the atherosclerotic lesion, with particular high mRNA levels in carotid plaques from patients with the most recent symptoms. Immunostaining of carotid plaques localized FABP4 to macrophages, while activated platelets and oxidized LDL were potent stimuli for FABP4 expression in monocytes/macrophages in vitro. When measured at the time of acute ischemic stroke, high plasma levels of FABP4 were significantly associated with total and cardiovascular mortality during follow-up, although we did not find that addition of FABP4 to the fully adjusted multivariate model had an effect on the prognostic discrimination for all-cause mortality as assessed by c-statistics.

Conclusions

FABP4 is linked to atherogenesis, plaque instability and adverse outcome in patients with carotid atherosclerosis and acute ischemic stroke.     

Metformin alleviates hepatosteatosis by restoring SIRT1-mediated autophagy induction via an AMP-activated protein kinase-independent pathway

Metformin activates both PRKA and SIRT1. Furthermore, autophagy is induced by either the PRKA-MTOR-ULK1 or SIRT1-FOXO signaling pathways. We aimed to elucidate the mechanism by which metformin alleviates hepatosteatosis by examining the molecular interplay between SIRT1, PRKA, and autophagy. ob/ob mice were divided into 3 groups: one with ad libitum feeding of a standard chow diet, one with 300 mg/kg intraperitoneal metformin injections, and one with 3 g/d caloric restriction (CR) for a period of 4 wk. Primary hepatocytes or HepG2 cells were treated with oleic acid (OA) plus high glucose in the absence or presence of metformin. Both CR and metformin significantly improved body weight and glucose homeostasis, along with hepatic steatosis, in ob/ob mice. Furthermore, CR and metformin both upregulated SIRT1 expression and also stimulated autophagy induction and flux in vivo. Metformin also prevented OA with high glucose-induced suppression of both SIRT1 expression and SIRT1-dependent activation of autophagy machinery, thereby alleviating intracellular lipid accumulation in vitro. Interestingly, metformin treatment upregulated SIRT1 expression and activated PRKA even after siRNA-mediated knockdown of PRKAA1/2 and SIRT1, respectively. Taken together, these results suggest that metformin alleviates hepatic steatosis through PRKA-independent, SIRT1-mediated effects on the autophagy machinery.     

Chlamydia pneumoniae exploits adipocyte lipid chaperone FABP4 to facilitate fat mobilization and intracellular growth in murine adipocytes

Fatty acid-binding protein 4 (FABP4), a cytosolic lipid chaperone predominantly expressed in adipocytes and macrophages, modulates lipid fluxes, trafficking, signaling, and metabolism. Recent studies have demonstrated that FABP4 regulates metabolic and inflammatory pathways, and in mouse models its inhibition can improve type 2 diabetes mellitus and atherosclerosis. However, the role of FABP4 in bacterial infection, metabolic crosstalk between host and pathogen, and bacterial pathogenesis have not been studied. As an obligate intracellular pathogen, Chlamydia pneumoniae needs to obtain nutrients such as ATP and lipids from host cells. Here, we show that C. pneumoniae successfully infects and proliferates in murine adipocytes by inducing hormone sensitive lipase (HSL)-mediated lipolysis. Chemical inhibition or genetic manipulation of HSL significantly abrogated the intracellular growth of C. pneumoniae in adipocytes. Liberated free fatty acids were utilized to generate ATP via β-oxidation, which C. pneumoniae usurped for its replication. Strikingly, chemical inhibition or genetic silencing of FABP4 significantly abrogated C. pneumoniae infection-induced lipolysis and mobilization of liberated FFAs, resulting in reduced bacterial growth in adipocytes. Collectively, these results demonstrate that C. pneumoniae exploits host FABP4 to facilitate fat mobilization and intracellular replication in adipocytes. This work uncovers a novel strategy used by intracellular pathogens for acquiring energy via hijacking of the host lipid metabolism pathway.     

Reduction of serum FABP4 level by sitagliptin,a DPP-4 inhibitor,in patients with type 2 diabetes mellitus

Fatty acid binding protein 4 (FABP4), also known as adipocyte FABP or aP2, is secreted from adipocytes in association with lipolysis as a novel adipokine, and elevated serum FABP4 level is associated with obesity, insulin resistance, and atherosclerosis. However, little is known about the modulation of serum FABP4 level by therapeutic drugs. Sitagliptin (50 mg/day), a dipeptidyl peptidase 4 (DPP-4) inhibitor that increases glucagon-like peptide 1 (GLP-1), was administered to patients with type 2 diabetes (n = 24) for 12 weeks. Treatment with sitagliptin decreased serum FABP4 concentration by 19.7% (17.8 ± 1.8 vs. 14.3 ± 1.5 ng/ml, P < 0.001) and hemoglobin A1c without significant changes in adiposity or lipid variables. In 3T3-L1 adipocytes, sitagliptin or exendin-4, a GLP-1 receptor agonist, had no effect on short-term (2 h) secretion of FABP4. However, gene expression and long-term (24 h) secretion of FABP4 were significantly reduced by sitagliptin, which was not mimicked by exendin-4. Treatment with recombinant DPP-4 increased gene expression and long-term secretion of FABP4, and the effects were cancelled by sitagliptin. Furthermore, knockdown of DPP-4 in 3T3-L1 adipocytes decreased gene expression and long-term secretion of FABP4. In conclusion, sitagliptin decreases serum FABP4 level, at least in part, via reduction in the expression and consecutive secretion of FABP4 in adipocytes by direct inhibition of DPP-4.     

Identification of new dual FABP4/5 inhibitors based on a naphthalene-1-sulfonamide FABP4 inhibitor

Fatty acid binding protein 4 (FABP4) and fatty acid binding protein 5 (FABP5) are mainly expressed in adipocytes and/or macrophages and play essential roles in energy metabolism and inflammation. When FABP4 function is diminished, FABP5 expression is highly increased possibly as a functional compensation. Dual FABP4/5 inhibitors are expected to provide beneficial synergistic effect on treating diabetes, atherosclerosis, and inflammation-related diseases. Starting from our previously reported selective FABP4 inhibitor 8, structural biology information was used to modulate the selectivity profile and to design potent dual FABP4/5 inhibitors with good selectivity against FABP3. Two compounds A16 and B8 were identified to show inhibitory activities against both FABP4/5 and good selectivity over FABP3, which could also reduce the level of forskolin-stimulated lipolysis in mature 3T3-L1 adipocytes. Compared with compound 8, these two compounds exhibited better anti-inflammatory effects in lipopolysaccharide-stimulated RAW264.7 murine macrophages, with decreased levels of pro-inflammatory cytokines TNFα and MCP-1 and apparently inhibited IKK/NF-κB pathway.     

Metformin reduces blood pressure and restores endothelial function in aorta of streptozotocin-induced diabetic rats

Effect of metformin treatment on blood pressure, endothelial function and oxidative stress in streptozotocin (STZ)-induced diabetes in rats was studied. In vitro effect of metformin on vascular reactivity to various agonist in the presence of metformin in untreated nondiabetic and STZ-diabetic rats were also studied. Sprague-Dawley rats were randomized into nondiabetic and STZ-diabetic groups. Rats were further randomized to receive metformin (150 mg/kg) or vehicle for 4 weeks.Metformin treatment reduced blood pressure without having any significant effect on blood glucose level in STZ-diabetic rats. Enhanced phenylephrine (PE)-induced contraction and impaired acetylcholine (Ach)-induced relaxation in STZ-diabetic rats were restored to normal by metformin treatment. Enhanced Ach-induced relaxation in metformin-treated STZ-diabetic rats was blocked due to pretreatment with 100 μM of Nω-nitro-l-arginine-methyl ester (l-NAME) or 10 μM of methylene blue but not 10 μM of indomethacin. Metformin treatment significantly increased antioxidant enzymes and reduced lipid peroxidation in STZ-diabetic rats. In vitro studies in aortic rings of untreated nondiabetic and STZ-diabetic rats showed that the presence of higher concentration of metformin (1 mM and 10 mM) significantly reduced PE-induced contraction and increased Ach-induced relaxation. Metformin per se relaxed precontracted aortic rings of untreated nondiabetic and STZ-diabetic rats in a dose-dependent manner. Pretreatment with l-NAME or removal of endothelium blocked metformin-induced relaxation at lower concentration (up to 30 μM) but not at higher concentration (above 30 μM). Metformin-induced relaxation was blocked in the presence of 1 mM of 4-aminopyridine, or 1 mM of tetraethylammonium but not in the presence of 100 μM of barium ion or 10 μM of glybenclamide. The restored endothelial function along with direct effect of metformin on aortic rings and reduced oxidative stress contributes to reduced blood pressure in STZ-diabetic rats. From the present study, it can be concluded that metformin administration to STZ-diabetic rats lowers blood pressure, and restores endothelial function.     

Metformin alleviates hepatosteatosis by restoring SIRT1-mediated autophagy induction via an AMP-activated protein kinase-independent pathway

Metformin activates both PRKA and SIRT1. Furthermore, autophagy is induced by either the PRKA-MTOR-ULK1 or SIRT1-FOXO signaling pathways. We aimed to elucidate the mechanism by which metformin alleviates hepatosteatosis by examining the molecular interplay between SIRT1, PRKA, and autophagy. ob/ob mice were divided into 3 groups: one with ad libitum feeding of a standard chow diet, one with 300 mg/kg intraperitoneal metformin injections, and one with 3 g/d caloric restriction (CR) for a period of 4 wk. Primary hepatocytes or HepG2 cells were treated with oleic acid (OA) plus high glucose in the absence or presence of metformin. Both CR and metformin significantly improved body weight and glucose homeostasis, along with hepatic steatosis, in ob/ob mice. Furthermore, CR and metformin both upregulated SIRT1 expression and also stimulated autophagy induction and flux in vivo. Metformin also prevented OA with high glucose-induced suppression of both SIRT1 expression and SIRT1-dependent activation of autophagy machinery, thereby alleviating intracellular lipid accumulation in vitro. Interestingly, metformin treatment upregulated SIRT1 expression and activated PRKA even after siRNA-mediated knockdown of PRKAA1/2 and SIRT1, respectively. Taken together, these results suggest that metformin alleviates hepatic steatosis through PRKA-independent, SIRT1-mediated effects on the autophagy machinery.