Apigenin targets fetuin-A to ameliorate obesity-induced insulin resistance.

Fetuin-A, a hepatokine secreted by hepatocytes, binds to insulin receptors and consequently impairs the activation of the insulin signaling pathway, leading to insulin resistance. Apigenin, a flavonoid isolated from plants, has beneficial effects on insulin resistance; however, its regulatory mechanisms are not fully understood. In the present study, we investigated the molecular mechanisms underlying the protective effects of apigenin on insulin resistance. In Huh7 cells, treatment with apigenin decreased the mRNA expression of fetuin-A by decreasing reactive oxygen species-mediated casein kinase 2α (CK2α)-nuclear factor kappa-light-chain-enhancer of activated B activation; besides, apigenin decreased the levels of CK2α-dependent fetuin-A phosphorylation and thus promoted fetuin-A degradation through the autophagic pathway, resulting in a decrease in the protein levels of fetuin-A. Moreover, apigenin prevented the formation of the fetuin-A-insulin receptor (IR) complex and thereby rescued the PA-induced impairment of the insulin signaling pathway, as evidenced by increased phosphorylation of IR substrate-1 and Akt, and translocation of glucose transporter 2 from the cytosol to the plasma membrane. Similar results were observed in the liver of HFD-fed mice treated with apigenin. Collectively, our findings revealed that apigenin ameliorates obesity-induced insulin resistance in the liver by targeting fetuin-A.

Ractopamine at legal residue dosage accelerates atherosclerosis by inducing endothelial dysfunction and promoting macrophage foam cell formation.

Ractopamine, a synthetic ß-adrenoreceptor agonist, is used as an animal feed additive to increase food conversion efficiency and accelerate lean mass accretion in farmed animals. The U.S. Food and Drug Administration claimed that ingesting products containing ractopamine residues at legal dosages might not cause short-term harm to human health. However, the effect of ractopamine on chronic inflammatory diseases and atherosclerosis is unclear. Therefore, we investigated the effects of ractopamine on atherosclerosis and its action mechanism in apolipoprotein E-null (apoe-/-) mice and human endothelial cells (ECs) and macrophages. Daily treatment with ractopamine for four weeks increased the body weight and the weight of brown adipose tissues and gastrocnemius muscles. However, it decreased the weight of white adipose tissues in apoe-/- mice. Additionally, ractopamine exacerbated hyperlipidemia and systemic inflammation, deregulated aortic cholesterol metabolism and inflammation, and accelerated atherosclerosis. In ECs, ractopamine treatment induced endothelial dysfunction and increased monocyte adhesion and transmigration across ECs. In macrophages, ractopamine dysregulated cholesterol metabolism by increasing oxidized low-density lipoprotein (oxLDL) internalization and decreasing reverse cholesterol transporters, increasing oxLDL-induced lipid accumulation. Collectively, our findings revealed that ractopamine induces EC dysfunction and deregulated cholesterol metabolism of macrophages, which ultimately accelerates atherosclerosis progression.

New Mechanisms of Bromelain in Alleviating Non-Alcoholic Fatty Liver Disease-Induced Deregulation of Blood Coagulation.

Bromelain, an enzyme extracted from the stems of pineapples, exerts anticoagulant effects; however, the regulatory mechanisms are not fully understood. Here, we aimed to investigate the effects of bromelain on non-alcoholic fatty liver disease (NAFLD)-induced deregulation of blood coagulation and the underlying molecular mechanisms. C57BL/6 mice were fed a high-fat diet (HFD), with or without bromelain (20 mg/kg/day) administration, for 12 weeks. Treatment with bromelain decreased thrombus formation in the liver and prolonged HFD-induced shortened prothrombin, activated partial thromboplastin, and fibrinogen times. Moreover, liquid chromatography-mass spectrometry/mass spectrometry and Western blot analysis showed that bromelain inhibited NAFLD-induced activation of the intrinsic, extrinsic, and common pathways by upregulating the protein expression of antithrombin III, serpin family G member 1, and α1-antitrypsin, and downregulating the protein expression of fibrinogen in the liver and plasma. Bromelain also upregulated the level of plasminogen and downregulating factor XIII expression in the liver and plasma. Collectively, these findings suggest that bromelain exerts anticoagulant effects on NAFLD-induced deregulation of coagulation by inhibiting the activation of the coagulation cascade, decreasing the stability of clots, and promoting fibrinolytic activity. The present study provides new insights into the potential therapeutic value of bromelain for the prevention and treatment of thrombosis-related diseases.

Genetic Deletion of HLJ1 Does Not Affect Blood Coagulation in Mice.

HLJ1 (also called DNAJB4) is a member of the DNAJ/Hsp40 family and plays an important role in regulating protein folding and activity. However, there is little information about the role of HLJ1 in the regulation of physiological function. In this study, we investigated the role of HLJ1 in blood coagulation using wild-type C57BL/6 mice and HLJ1-null (HLJ1-/-) mice. Western blot analysis and immunohistochemistry were used to assess the expression and distribution of HLJ1 protein, respectively. The tail bleeding assay was applied to assess the bleeding time and blood loss. A coagulation test was used for measuring the activity of extrinsic, intrinsic and common coagulation pathways. Thromboelastography was used to measure the coagulation parameters in the progression of blood clot formation. The results showed that HLJ1 was detectable in plasma and bone marrow. The distribution of HLJ1 was co-localized with CD41, the marker of platelets and megakaryocytes. However, genetic deletion of HLJ1 did not alter blood loss and the activity of extrinsic and intrinsic coagulation pathways, as well as blood clot formation, compared to wild-type mice. Collectively, these findings suggest that, although HLJ1 appears in megakaryocytes and platelets, it may not play a role in the function of blood coagulation under normal physiological conditions.

Bromelain Ameliorates Atherosclerosis by Activating the TFEB-Mediated Autophagy and Antioxidant Pathways.

Bromelain, a cysteine protease found in pineapple, has beneficial effects in the treatment of inflammatory diseases; however, its effects in cardiovascular pathophysiology are not fully understood. We investigated the effect of bromelain on atherosclerosis and its regulatory mechanisms in hyperlipidemia and atheroprone apolipoprotein E-null (apoe-/-) mice. Bromelain was orally administered to 16-week-old male apoe-/- mice for four weeks. Daily bromelain administration decreased hyperlipidemia and aortic inflammation, leading to atherosclerosis retardation in apoe-/- mice. Moreover, hepatic lipid accumulation was decreased by the promotion of cholesteryl ester hydrolysis and autophagy through the AMP-activated protein kinase (AMPK)/transcription factor EB (TFEB)-mediated upregulation of autophagy- and antioxidant-related proteins. Moreover, bromelain decreased oxidative stress by increasing the antioxidant capacity and protein expression of antioxidant proteins while downregulating the protein expression of NADPH oxidases and decreasing the production of reactive oxygen species. Therefore, AMPK/TFEB signaling may be crucial in bromelain-mediated anti-hyperlipidemia, antioxidant, and anti-inflammatory effects, effecting the amelioration of atherosclerosis.

Apigenin ameliorates hepatic lipid accumulation by activating the autophagy-mitochondria pathway.

Apigenin, a flavonoid isolated from plants, provides protection against non-alcoholic fatty liver disease. However, the mechanism by which apigenin decreases lipid accumulation in the liver is unclear. In this study, we investigated the molecular mechanism underlying the beneficial effect of apigenin on the hepatic deregulation of lipid metabolism. Oleic acid (OA)-induced lipid accumulation in human hepatoma cells (Huh7 cells) was used as an in vitro model. Western blot analysis was used for evaluating protein expression. Oil red O staining, Nile red staining, and conventional assay kits were used to assess the level of lipids. Immunocytochemistry was performed to observe mitochondrial morphology. Seahorse XF analyzer was used to measure mitochondrial bioenergetics. Treatment with OA induced lipid accumulation in Huh7 cells, which was attenuated by apigenin. Mechanistically, treatment with apigenin increased the expression of autophagy-related proteins including Beclin1, autophagy related gene 5 (ATG5), ATG7, and LC3II, and the formation of autophagolysosomes, leading to an increase in intracellular levels of fatty acids. Inhibition of autophagy by bafilomycin A1 or chloroquine abolished the protection of apigenin in OA-induced lipid accumulation. Apigenin up-regulated the protein expression related to the ß-oxidation pathway including acyl-CoA synthetase long chain family member 1, carnitine palmitoyltransferase 1α, acyl-CoA oxidase 1, peroxisome proliferator activated receptor (PPAR) α, and PPARγ coactivator 1-α. Moreover, apigenin increased the mitochondrial network structure and mitochondrial function by increasing the protein expression related to the process of mitochondria fusion and mitochondrial function. Collectively, our findings suggest that apigenin ameliorates hepatic lipid accumulation by activating the autophagy-mitochondrial pathway.

Bromelain Confers Protection against the Non-Alcoholic Fatty Liver Disease in Male C57bl/6 Mice.

We aimed to investigate the effect of bromelain, the extract from stems of pineapples on the high-fat diet (HFD)-induced deregulation of hepatic lipid metabolism and non-alcoholic fatty liver disease (NAFLD), and its underlying mechanism in mice. Mice were daily administrated with HFD with or without bromelain (20 mg/kg) for 12 weeks, and we found that bromelain decreased the HFD-induced increase in body weight by ~30%, organ weight by ~20% in liver weight and ~40% in white adipose tissue weight. Additionally, bromelain attenuated HFD-induced hyperlipidemia by decreasing the serum level of total cholesterol by ~15% and triglycerides level by ~25% in mice. Moreover, hepatic lipid accumulation, particularly that of total cholesterol, free cholesterol, triglycerides, fatty acids, and glycerol, was decreased by 15-30% with bromelain treatment. Mechanistically, these beneficial effects of bromelain on HFD-induced hyperlipidemia and hepatic lipid accumulation may be attributed to the decreased fatty acid uptake and cholesteryl ester synthesis and the increased lipoprotein internalization, bile acid metabolism, cholesterol clearance, the assembly and secretion of very low-density lipoprotein, and the ß-oxidation of fatty acids by regulating the protein expression involved in the above mentioned hepatic metabolic pathways. Collectively, these findings suggest that bromelain has therapeutic value for treating NAFLD and metabolic diseases.

Food preservative sorbic acid deregulates hepatic fatty acid metabolism.

Sorbic acid (SA) is one of the most commonly used food preservatives worldwide. Despite SA having no hepatotoxicity at legal dosages, its effect on hepatic lipid metabolism is still unclear. We investigated the effect of SA on hepatic lipid metabolism and its mechanism of action in C57BL/6 mice. Daily treatment with SA (1 g/kg in diet) for 4 weeks did not alter the body weight, organ weight, and blood lipids in mice. However, hepatic lipid accumulation, particularly that of triglycerides, fatty acids, and glycerol, but not cholesteryl ester and free cholesterol, was increased with SA treatment. Mechanistically, SA decreased the expression of proteins related to de novo fatty acid lipogenesis, fatty acid internalization, and very low-density lipoprotein (VLDL) secretion-related pathways, including sterol regulatory element-binding proteins, acetyl-coA carboxylase, fatty acid synthase, liver fatty acid-binding protein, CD36, and apolipoprotein E. In contrast, SA increased the expression of diacylglycerol O-acyltransferase 2, the key enzyme for triacylglycerol synthesis. Moreover, SA downregulated the protein expression of autophagy-related and ß-oxidation-related pathways, the two major metabolic pathways for lipid metabolism, including LC-3, beclin-1, autophagy related protein 5 (ATG-5) and ATG-7, acyl-CoA synthetase long chain family member 1, carnitine palmitoyltransferase Iα, peroxisome proliferator-activated receptor α (PPARα), PPARγ, and PPARγ coactivator-1. Collectively, SA deregulates de novo lipogenesis and fatty acid internalization, VLDL secretion, autophagy, and ß-oxidation in the liver, leading to impaired lipid clearance and ultimately, resulting in lipid accumulation in the liver.