Kruppel-like factor 14 ameliorated obesity and related metabolic disorders by promoting adipose tissue browning.

Obesity has been identified as a serious and debilitating disease that threatens human health, but the current treatment strategies still have some shortcomings. Exercise and dieting are difficult for many people to adhere to, and a series of surgical risks and pain brought about by volume reduction have made it difficult for the current weight loss effect to meet human expectations. In this study, we first found that mice with overexpression of the transcription factor Kruppel-like factor 14 (KLF14) in subcutaneous adipose tissue gained weight more slowly while consuming a high-fat diet than did control mice, and these mice also showed reduced insulin resistance and liver lipid deposition abnormalities. Mechanistically, the browning of white adipose tissue was promoted in adipose tissue with KLF14 overexpression; therefore, we preliminarily concluded that KLF14 can improve obesity by promoting the browning of white adipose tissue and energy consumption, thus ameliorating obesity and related metabolic disturbances. In summary, our results revealed that KLF14 may promote white adipose tissue browning, thus ameliorating high-fat diet-induced obesity and hepatic steatosis, as well as serum lipid levels and insulin resistance, thereby achieving a positive effect on metabolism.NEW & NOTEWORTHY Our study first explored the role of KLF14 in the development and progression of HFD-induced obesity in male mice. Its beneficial effect on adipose browning and metabolic disorders suggests that KLF14 may provide us a new therapeutic strategy for obesity and related metabolic complications. This health problem is of global concern and needs to be addressed.

Inhibition of NFAT suppresses foam cell formation and the development of diet-induced atherosclerosis.

Deciphering the molecular and cellular processes involved in foam cell formation is critical for us to understand the pathogenesis of atherosclerosis. Nuclear factor of activated T cells (NFAT) is a transcription factor originally identified as a key player in the differentiation of T cells and maturation of immune system. Nowadays it has been brought into attention that NFAT also regulates multiple pathophysiological processes and targeted intervention in NFAT may be effective in the treatment of some cardiovascular diseases. However, whether NFAT is involved in foam cell formation remains elusive. NFAT in human monocyte-derived macrophage was activated by ox-LDL and translocated from the cytoplasm to the nucleus. NFAT then directly bound to peroxisome proliferator-activated receptor γ (PPARγ) in the nucleus and negatively regulated its transcriptional activity. NFATc2 knockdown or NFAT inhibitor 11R-VIVIT increased cholesterol efflux (by activating PPARγ-LXRα-ABCA1 cascade) and reduced the uptake of modified lipoprotein (in a PPARγ-independent way) in macrophage, thus prevented foam cell formation. Besides, 11R-VIVIT also exerted a protective role in the development of atherosclerosis in western diet-fed ApoE-/- mice. These results suggest NFAT inhibition as a potential therapeutic strategy in atherosclerosis.

Enoyl coenzyme A hydratase 1 alleviates nonalcoholic steatohepatitis in mice by suppressing hepatic ferroptosis.

Nonalcoholic steatohepatitis (NASH) is a common metabolic disorder that is a major contributor to health care expenditures worldwide. Enoyl coenzyme A hydratase 1 (ECH1) is initially recognized as a key component in mitochondrial fatty acid ß-oxidation, and subsequent studies have demonstrated that it regulates multiple pathophysiological processes. However, the relationship between ECH1 and NASH has remained largely unknown. Herein, we investigated the role of ECH1 in NASH progression. Adeno-associated virus-mediated genetic engineering was used to investigate the role of ECH1. Alterations in hepatic steatosis, inflammation, fibrogenesis, oxidative stress, apoptosis, and liver injury were monitored using liver or serum samples from mice. ECH1 expression was significantly higher in human NASH biopsy specimens and in methionine choline-deficient (MCD) diet-fed mice. ECH1 overexpression significantly alleviated hepatic steatosis, inflammation, fibrogenesis, apoptosis, and oxidative stress in livers of mice. In addition, ECH1 overexpression also reduced alanine aminotransferase and proinflammatory cytokine levels in serum and triglyceride levels in livers. Consistently, ECH1 knockdown suppressed this beneficial phenotype. Mechanistically, ECH1-knockdown mice treated with ferrostatin-1 (Fer-1) showed an alleviated NASH phenotype compared with the untreated knockdown mice. Meanwhile, we detected changes in Erk signaling pathway when ECH1 was overexpressed or knocked down, which may partially explain the potential mechanism of ECH1 regulation of ferroptosis.In summary, ECH1 may ameliorate steatohepatitis by inhibiting ferroptosis. Pharmacological or genetic ECH1 activation may have potential as a future therapy for NASH.NEW & NOTEWORTHY Enoyl coenzyme A hydratase 1 (ECH1) is a key component in mitochondrial fatty acid ß-oxidation and is also a well-known enzyme for lipid metabolism. However, the biological role of ECH1 in the development of NASH is still unclear. Herein, we demonstrated that ECH1 inhibits NASH by inhibiting ferroptosis, thus providing a novel target for therapeutic intervention for future treatment of NASH.

Targeting NFATc4 attenuates non-alcoholic steatohepatitis in mice.

BACKGROUND & AIMS: The nuclear factor of activated T-cells (NFAT) family was first recognised to play an important role in the differentiation of T cells, but has since been shown to regulate multiple pathophysiological processes. However, whether it is involved in the pathogenesis of non-alcoholic steatohepatitis (NASH) remains unknown. METHODS: Hepatic NFATc expression and localisation were analysed in C57BL/6 mice on a methionine-choline-deficient diet, as well as in samples from non-alcoholic fatty liver disease patients. Gain- or loss-of-function approaches were used to investigate the role of NFATc4 in NASH. RESULTS: NFATc4 translocates from the cytoplasm to the nucleus in hepatocytes of both humans and rodents with NASH. NFATc4 knockdown resulted in decreased hepatic steatosis, inflammation, and fibrosis during NASH progression. Mechanistically, we found that activated NFATc4 directly bound to peroxisome proliferator-activated receptor α (PPARα) in the nucleus and negatively regulated its transcriptional activity, thereby impairing the hepatic fatty acid oxidation pathway and increasing lipid deposition in the liver. Moreover, NFATc4 activation increased the production and secretion of osteopontin (OPN) from hepatocytes, which subsequently enhanced the macrophage-mediated inflammatory response and hepatic stellate cell-mediated fibrosis progression via paracrine signalling. CONCLUSIONS: Hepatic NFATc4 activation accelerates the progression of NASH by suppressing PPARα signalling and increasing OPN expression. Genetic or pharmacological inhibition of NFATc4 may have potential for future therapy of NASH. LAY SUMMARY: NFATc4 is activated in the non-alcoholic steatohepatitis of mice and patients. Inhibition of NFATc4 activation alleviates lipid deposition, inflammatory response, and fibrosis progression in the liver.

Enoyl coenzyme A hydratase 1 combats obesity and related metabolic disorders by promoting adipose tissue browning.

Browning of white adipose tissue (WAT) has been recognized as an important strategy for the treatment of obesity, insulin resistance, and diabetes. Enoyl coenzyme A hydratase 1 (ECH1) is a widely known enzyme involved in lipid metabolism. However, whether and how ECH1 is implicated in browning of WAT remain obscure. Adeno-associated, virus-mediated genetic engineering of ECH1 in adipose tissue was used in investigations in mouse models of obesity induced by a high-fat diet (HFD) or browning induced by cold exposure. Metabolic parameters showed that ECH1 overexpression decreased weight gain and improved insulin sensitivity and lipid profile after 8 wk of an HFD. Further work revealed that these changes were associated with enhanced energy expenditure and increased appearance of brown-like adipocytes in inguinal WAT, as verified by a remarkable increase in uncoupling protein 1 and thermogenic gene expression. In vitro, ECH1 induced brown fat-related gene expression in adipocytes differentiated from primary stromal vascular fractions, whereas knockdown of ECH1 reversed this effect. Mechanistically, ECH1 regulated the thermogenic program by inhibiting mammalian target of rapamycin signaling, which may partially explain the potential mechanism for ECH1 regulating adipose browning. In summary, ECH1 may participate in the pathology of obesity by regulating browning of WAT, which probably provides us with a new therapeutic strategy for combating obesity.

Role of adipokine zinc-α2-glycoprotein in coronary heart disease.

Zinc-α2-glycoprotein (AZGP1) is a newly identified adipokine that is associated with lipid metabolism and vascular fibrosis. Although adipokines contribute to lipid dysfunction and its related diseases, including stroke and coronary heart disease (CHD), the role of AZGP1 remains unclear. In this study, the role of AZGP1 in atherosclerosis and CHD was investigated. Serum AZGP1 levels from control (n = 84) and CHD (n = 91) patients were examined by ELISA and its relationship with various clinical parameters was analyzed. Immunohistochemistry and immunofluorescence were used to detect the expression of AZGP1 and its receptor in coronary atherosclerotic arteries. THP-1 and human embryonic kidney 293 cells were used to verify its anti-inflammatory role in atherosclerosis. Serum AZGP1 levels in CHD patients were lower than controls (P < 0.01) and independently associated with CHD prevalence (P = 0.021). AZGP1 levels also inversely correlated with the Gensini score. Immunohistochemistry and immunofluorescence showed that AZGP1 and its receptor ß3-adrenoceptor (ß3-AR) colocalized in lipid-rich areas of atherosclerotic plaques, particularly around macrophages. In vitro, AZGP1 had no effect on foam cell formation but showed anti-inflammatory effects through its regulation of JNK/AP-1 signaling. In summary, AZGP1 is an anti-inflammatory agent that can be targeted for CHD treatment.

Absence of Interferon Regulatory Factor 1 Protects Against Atherosclerosis in Apolipoprotein E-Deficient Mice.

Deciphering the molecular and cellular processes involved in foam cell formation is critical to understanding the pathogenesis of atherosclerosis. Interferon regulatory factor 1 (IRF1) was first identified as a transcriptional regulator of type-I interferons (IFNs) and IFN inducible genes. Our study aims to explore the role of IRF1 in atherosclerotic foam cell formation and understand the functional diversity of IRF1 in various cell types contributing to atherosclerosis. Methods: We induced experimental atherosclerosis in ApoE-/-IRF1-/- mice and evaluated the effect of IRF1 on disease progression and foam cell formation. Results: IRF1 expression was increased in human and mouse atherosclerotic lesions. IRF1 deficiency inhibited modified lipoprotein uptake and promoted cholesterol efflux, along with altered expression of genes implicated in lipid metabolism. Gene expression analysis identified scavenger receptor (SR)-AI as a regulated target of IRF1, and SR-AI silencing completely abrogated the increased uptake of modified lipoprotein induced by IRF1. Our data also explain a mechanism underlying endotoxemia-complicated atherogenesis as follows: two likely pro-inflammatory agents, oxidized low-density lipoprotein (ox-LDL) and bacterial lipopolysaccharide (LPS), exert cooperative effects on foam cell formation, which is partly attributable to a shift of IRF1-Ubc9 complex to IRF1- myeloid differentiation primary response protein 88 (Myd88) complex and subsequent IRF1 nuclear translocation. Additionally, it seems that improved function of vascular smooth muscle cells (VSMCs) also accounts for the diminished and more stable atherosclerotic plaques observed in ApoE-/-IRF1-/- mice. Conclusions: Our findings demonstrate an unanticipated role of IRF1 in the regulation of gene expression implicated in foam cell formation and identify IRF1 activation as a new risk factor in the development, progression and instability of atherosclerotic lesions.

Role of CCR2 in the Development of Streptozotocin-Treated Diabetic Cardiomyopathy.

CCR2 has been proven to play an important role in diabetes. However, the role of CCR2 in diabetic cardiomyopathy has not been examined. In this study, we investigated the effects of cardiac CCR2 on diabetic cardiomyopathy. We created a model of streptozotocin (STZ)-induced diabetic cardiomyopathy. Expression of CCR2 was upregulated in the hearts of STZ-induced diabetic mice. CCR2 knockout significantly improved STZ-induced cardiac dysfunction and fibrosis. Moreover, deletion of CCR2 inhibited STZ-induced apoptosis and the production of STZ-induced reactive oxygen species in the heart. CCR2 knockout resulted in M2 polarization in hearts of STZ-treated mice. Treatment with a CCR2 inhibitor reversed hyperglycemia-induced cardiac dysfunction in db/db mice. These results suggest that CCR2-induced inflammation and oxidative stress in the heart are involved in the development of diabetic cardiomyopathy and that CCR2 could be a novel target for therapy.

Novel role of NLRP3-inflammasome in regulation of lipogenesis in fasting-induced hepatic steatosis.

Background: The liver coordinates a series of metabolic adaptations to maintain the energy balance of the system and provide adequate nutrients to key organs, tissues and cells during starvation. However, the mediators and underlying molecular mechanisms that mediate these fasting-induced adaptive responses remain unclear. Materials and methods: Male wild-type C57BL/6J littermates (8-weeks-old) were intraperitoneally injected with MCC950 or vehicle, and then randomly divided into three groups: fed, fasted, and refed. Plasma IL1ß and insulin levels were detected by ELISA kits. Plasma and hepatic metabolites were determined using commercial assay kits. HepaRG cell line was applied to verify the regulation of NLRP3 on lipogenesis. Results: NOD-like receptor protein 3 (NLRP3) and its downstream inflammatory cytokines were significantly suppressed after 24 h fasting and recovered upon 6 h refeeding in plasma and liver tissues of mice. Moreover, fasting-induced hepatic steatosis and accompanied liver injury were ameliorated when mice were intraperitoneally injected with MCC950 (a selective NLRP3 inhibitor). Further study revealed that MCC950 suppressed sterol regulatory element-binding protein-1c (SREBP-1c) expression and transcriptional activity, thus inhibited lipogenesis in the liver, which may explain its role in stabilizing lipid metabolism. Conclusion: The NLRP3 inhibitor-MCC950 protects against fasting-induced hepatic steatosis. The novel and critical role of NLRP3 in lipogenesis may explain its importance in regulating the adaptive responses of the liver upon starvation stress and may provide therapeutic value.

Poly(ADP-ribose) Polymerase-1 is required for hepatocyte proliferation and liver regeneration in mice.

Liver regeneration is an orchestrated cellular response and the mechanisms underlying it have been extensively studied using the partial hepatectomy (PHx) model. Poly(ADP-Ribose) Polymerase-1 (PARP1) is a common enzyme for post-translational modification of proteins. Here, we aimed to determine the role of PARP1 in liver regeneration. We found that PARP1 activity was strongly associated with hepatocyte proliferation after PHx. PARP1 knockout mice showed impaired liver recovery and suppressed hepatocyte proliferation after PHx. Mechanistically, PARP1 knockout repressed YAP activity and inhibited expression of cell cycle-associated proteins in liver tissues. Therefore, our findings highlight specialized roles for PARP1 in regulating hepatocytes proliferation and liver regeneration.

The novel adipokine CTRP5 is a negative regulator of white adipose tissue browning.

The browning of white adipose tissue predominantly emerges as an adaptation to environmental cues, such as cold exposure. The enhanced browning of adipose tissue results in improved energy and glucose homeostasis and reduced fat mass and body weight, which is greatly beneficial for the treatment of obesity and other metabolic diseases. C1q/TNF-related protein 5 (CTRP5) is a novel adipokine associated with a variety of endocrine and metabolic diseases; however, whether it can regulate the metabolism of adipose tissue itself remains unknown. In this study, we demonstrated that the expression of CTRP5 in murine subcutaneous white adipose tissue (scWAT) was significantly decreased when the mice were exposed to cold temperatures. The lentivirus-mediated overexpression of CTRP5 in mice repressed the adipose tissue browning, leading to reduced heat production, decreased expression of the browning marker uncoupling protein 1 (UCP1) and decreased browning-related gene expression. Mechanistically, we found that autophagy was inhibited after cold exposure, but this inhibition was alleviated after CTRP5 overexpression. In primary cultured adipocytes, CTRP5 suppressed UCP1 expression, whereas 3-MA (an autophagy inhibitor) rescued the suppression. All of these results demonstrated that CTRP5 is a negative regulator of adipose browning. CTRP5 exerts its effect, at least in part, by suppressing adipocyte autophagy. Our findings indicated that CTRP5 is a novel promising therapeutic target for obesity and other metabolic diseases.

Hyperglycemia-Driven Inhibition of AMP-Activated Protein Kinase α2 Induces Diabetic Cardiomyopathy by Promoting Mitochondria-Associated Endoplasmic Reticulum Membranes In Vivo.

BACKGROUND: Fundc1 (FUN14 domain containing 1), an outer mitochondrial membrane protein, is important for mitophagy and mitochondria-associated endoplasmic reticulum membranes (MAMs). The roles of Fundc1 and MAMs in diabetic hearts remain unknown. The aims of this study, therefore, were to determine whether the diabetes mellitus-induced Fundc1 expression could increase MAM formation, and whether disruption of MAM formation improves diabetic cardiac function. METHODS: Levels of FUNDC1 were examined in the hearts from diabetic patients and nondiabetic donors. Levels of Fundc1-induced MAMs and mitochondrial and heart function were examined in mouse neonatal cardiomyocytes exposed to high glucose (HG, 30 mmol/L d-glucose for 48 hours), and in streptozotocin-treated cardiac-specific Fundc1 knockout mice and cardiac-specific Fundc1 knockout diabetic Akita mice, as well. RESULTS: FUNDC1 levels were significantly elevated in cardiac tissues from diabetic patients in comparison with those from nondiabetic donors. In cultured mouse neonatal cardiomyocytes, HG conditions increased levels of Fundc1, the inositol 1,4,5-trisphosphate type 2 receptor (Ip3r2), and MAMs. Genetic downregulation of either Fundc1 or Ip3r2 inhibited MAM formation, reduced endoplasmic reticulum-mitochondrial Ca2+ flux, and improved mitochondrial function in HG-treated cardiomyocytes. Consistently, adenoviral overexpression of Fundc1 promoted MAM formation, mitochondrial Ca2+ increase, and mitochondrial dysfunction in cardiomyocytes exposed to normal glucose (5.5 mmol/L d-glucose). In comparison with nondiabetic controls, levels of Fundc1, Ip3r2, and MAMs were significantly increased in hearts from streptozotocin-treated mice and Akita mice. Furthermore, in comparison with control hearts, diabetes mellitus markedly increased coimmunoprecipitation of Fundc1 and Ip3r2. The binding of Fundc1 to Ip3r2 inhibits Ip3r2 ubiquitination and proteasome-mediated degradation. Cardiomyocyte-specific Fundc1 deletion ablated diabetes mellitus-induced MAM formation, prevented mitochondrial Ca2+ increase, mitochondrial fragmentation, and apoptosis with improved mitochondrial functional capacity and cardiac function. In mouse neonatal cardiomyocytes, HG suppressed AMP-activated protein kinase activity. Furthermore, in cardiomyocytes of Prkaa2 knockout mice, expression of Fundc1, MAM formation, and mitochondrial Ca2+ levels were significantly increased. Finally, adenoviral overexpression of a constitutively active mutant AMP-activated protein kinase ablated HG-induced MAM formation and mitochondrial dysfunction. CONCLUSIONS: We conclude that diabetes mellitus suppresses AMP-activated protein kinase, initiating Fundc1-mediated MAM formation, mitochondrial dysfunction, and cardiomyopathy, suggesting that AMP-activated protein kinase-induced Fundc1 suppression is a valid target to treat diabetic cardiomyopathy.

The LPS-inducible lncRNA Mirt2 is a negative regulator of inflammation.

Toll-like receptors (TLRs) are a family of pattern recognition receptors (PRR) with a crucial function in innate immune responses. Activation of TLR4 signaling at the plasma membrane by lipopolysaccharide (LPS) stimulates proinflammatory signaling pathways dependent on the E3 ubiquitin ligase TRAF6. Here we show the LPS-induced long non-coding RNA (lncRNA) Mirt2 functions as a checkpoint to prevent aberrant activation of inflammation, and is a potential regulator of macrophage polarization. Mirt2 associates with, and attenuates Lys63 (K63)-linked ubiquitination of, TRAF6, thus inhibiting activation of NF-κB and MAPK pathways and limiting production of proinflammatory cytokines. Adenovirus mediated gene transfer of Mirt2 protects mice from endotoxemia induced fatality and multi-organ dysfunction. These findings identify lncRNA Mirt2 as a negative feedback regulator of excessive inflammation.

Binding of FUN14 Domain Containing 1 With Inositol 1,4,5-Trisphosphate Receptor in Mitochondria-Associated Endoplasmic Reticulum Membranes Maintains Mitochondrial Dynamics and Function in Hearts in Vivo.

BACKGROUND: FUN14 domain containing 1 (FUNDC1) is a highly conserved outer mitochondrial membrane protein. The aim of this study is to examine whether FUNDC1 modulates the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), mitochondrial morphology, and function in cardiomyocytes and intact hearts. METHODS: The impacts of FUNDC1 on MAMs formation and cardiac functions were studied in mouse neonatal cardiomyocytes, in mice with cardiomyocyte-specific Fundc1 gene knockout (Fundc1f/Y/CreαMyHC+/- ), and in the cardiac tissues of the patients with heart failure. RESULTS: In mouse neonatal cardiomyocytes and intact hearts, FUNDC1 was localized in MAMs by binding to ER-resided inositol 1,4,5-trisphosphate type 2 receptor (IP3R2). Fundc1 ablation disrupted MAMs and reduced the levels of IP3R2 and Ca2+ in both mitochondria and cytosol, whereas overexpression of Fundc1 increased the levels of IP3R2 and Ca2+ in both mitochondria and cytosol. Consistently, Fundc1 ablation increased Ca2+ levels in ER, whereas Fundc1 overexpression lowered ER Ca2+ levels. Further, Fundc1 ablation in cardiomyocytes elongated mitochondria and compromised mitochondrial functions. Mechanistically, we found that Fundc1 ablation-induced reduction of intracellular Ca2+ levels suppressed mitochondrial fission 1 protein (Fis1) expression and mitochondrial fission by reducing the binding of the cAMP response element binding protein (CREB) in the Fis1 promoter. Fundc1f/Y/CreαMyHC+/- mice but not their littermate control mice (Fundc1wt/Y/CreαMyHC+/- ) exhibited cardiac dysfunction. The ligation of the left ventricle artery of Fundc1f/Y/CreαMyHC+/- mice caused more severe cardiac dysfunction than those in sham-treated Fundc1f/Y/CreαMyHC+/- mice. Finally, we found that the FUNDC1/MAMs/CREB/Fis1 signaling axis was significantly suppressed in patients with heart failure. CONCLUSIONS: We conclude that FUNDC1 binds to IP3R2 to modulate ER Ca2+ release into mitochondria and cytosol. Further, a disruption of the FUNDC1 and IP3R2 interaction lowers the levels of Ca2+ in mitochondria and cytosol, both of which instigate aberrant mitochondrial fission, mitochondrial dysfunction, cardiac dysfunction, and heart failure.