Raspberry ketone promotes FNDC5 protein expression via HO-1 upregulation in 3T3-L1 adipocytes.

Obesity is a global health problem and a risk factor for cardiovascular diseases and cancers. Exercise is an effective intervention to combat obesity. Fibronectin type III domain containing protein 5 (FNDC5)/irisin, a myokine, can stimulate the browning of white adipose tissue by increasing uncoupling protein 1 (UCP1) expression, and therefore may represent a link between the beneficial effects of exercise and improvement in metabolic diseases. Thus, upregulating the endogenous expression of FNDC5/irisin by administering medication would be a good approach for treating obesity. Herein, we evaluated the efficacy of raspberry ketone (RK) in inducing FNDC5/irisin expression and the underlying mechanisms. The expression of brown fat-specific proteins (PR domain containing 16 (PRDM16), CD137, and UCP1), heme oxygenase-1 (HO-1), FNDC5, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) in differentiated 3T3-L1 adipocyte was analyzed by western blotting or immunofluorescence. The level of irisin in the culture medium was also assayed using an enzyme-linked immunosorbent assay kit. Results showed that RK (50 µM) significantly induced the upregulation of FNDC5 protein in differentiated 3T3-L1 adipocytes; however, the irisin level in the culture media was unaffected. Moreover, RK significantly increased the levels of PGC1α, brown adipocyte markers (PRDM16, CD137, and UCP1), and HO-1. Furthermore, the upregulation of PGC1α and FNDC5 and the browning effect induced by RK were significantly reduced by SnPP or FNDC5 siRNA, respectively. In conclusion, RK can induce FNDC5 protein expression via the HO-1 signaling pathway, and this study provides new evidence for the potential use of RK in the treatment of obesity.

Ethyl pyruvate ameliorates heat stroke-induced multiple organ dysfunction and inflammatory responses by induction of stress proteins and activation of autophagy in rats.

Objective: Heat stroke (HS) elicits the systemic inflammatory responses that result in multiple organ dysfunction (MOD). Heat shock response and autophagy are activated during heat stress for removal of damaged organelles and proteins, emerging as a major regulator of cellular homeostasis. Ethyl pyruvate (EP) is a derivative of pyruvic acid and possesses antioxidant and anti-inflammatory effects. This study aims to investigate the effects of EP on MOD in HS rats and explore the possible mechanisms.Method: Anesthetized rats were placed in a heating chamber (42 °C) to elevate the core body temperature attaining to 42.9 °C. Rats were then moved to room temperature and monitored for 6 h. EP (60 mg/kg, i.v.) was administered 30 min prior to heat exposure.Results: Results showed that EP significantly reduced HS-induced increases in plasma levels of LDH, CPK, GPT and CK-MB, reversed the decrease of platelet counts, and alleviated intestinal mucosal and pulmonary damage. Moreover, EP reduced pro-inflammatory protein, including TNF-α, IL-6, IL-1ß, HMGB1 and iNOS, and induced stress proteins, heme oxygenase-1 (HO-1), heat shock protein (HSP) 70 and HSP90 in the liver of HS rats. The levels of HS-activated autophagy-regulatory proteins were affected by EP, in which the phosphorylated mTOR and AKT were reduced, and the phosphorylated AMPK increased, accompanied with upregulation in ULK1, Atg7, Atg12 and LC3II, and downregulation of p62.Conclusion: In conclusion, EP ameliorated HS-induced inflammatory responses and MOD, and the underlying mechanism is associated with the induction of the stress proteins HO-1 and HSP70 as well as restorage of autophagy.

Raloxifene inhibits adipose tissue inflammation and adipogenesis through Wnt regulation in ovariectomized rats and 3 T3-L1 cells.

BACKGROUND: Loss of ovarian function, as in menopause or after ovariectomy (OVX), is closely associated with obesity and white adipose tissue (WAT) inflammation. Estrogen replacement protects against postmenopausal obesity but increases the risks of carcinogenesis. In the present study, we investigated the effects of long-term treatment of raloxifene (RAL), a selective estrogen receptor modulator, on the features of estrogen deficiency-induced obesity and explored the involvement of canonical and non-canonical Wnt regulation in vivo and in vitro. METHODS: Adult female rats received bilateral OVX and divided into 5 groups: (1) Sham, (2) OVX, (3) OVX + E2: OVX rats were administered with E2 (50 µg/kg, s.c., 3 times/week), (4) OVX + RAL: OVX rats were treated with RAL (gavage, 1 mg/kg/day) suspended in 0.8% carboxymethylcellulose (CMC), (5) OVX + CMC: 0.8% CMC as vehicle control. All treatments were given for 8 weeks beginning at 1 week after OVX. In 3 T3-L1 cells, the effects of RAL on adipogenesis and lipopolysaccharide (LPS)-induced inflammation were evaluated. RESULTS: Treatment with RAL significantly decreased body weight, visceral fat pad mass, adipocyte size and plasma levels of glucose but increased plasma adiponectin. RAL reduced the elevation of HIF-1α, VEGF-A and proinflammatory cytokines (MCP-1 and TNF-α) expression by inhibition of NF-κB p65 and JNK cascades in retroperitoneal WAT. This anti-inflammatory capacity of RAL may result from upregulation of secreted frizzle-related protein 5 (SFRP5), an adipokine that repressed Wnt5a signaling. Furthermore, RAL inhibited adipogenic factors such as PPAR-γ, C/EBP-α, and FABP4, and preserved canonical Wnt10b/ß-catenin protein expression. In 3 T3-L1 adipocytes, RAL (20 µM) diminished lipid accumulation and inhibited adipogenic factors accompanied with the induction of ß-catenin, which were effectively reversed by the ß-catenin inhibitor IWR-1-endo. In addition, RAL reduced LPS-induced NF-κB p65 and p-IκB expression as well as TNF-α secretion. Suppression of SFRP5 by small interfering RNA significantly abrogated the anti-inflammatory effects of RAL. CONCLUSIONS: Distinct activation of canonical ß-catenin on inhibition of adipogenesis and non-canonical SFRP5 on suppression of WAT inflammation may contribute to the beneficial effects of RAL. Therefore, this study provides a rationale for the therapeutic potential of RAL for postmenopausal obesity.

Alpha-Lipoic Acid Protects Cardiomyocytes against Heat Stroke-Induced Apoptosis and Inflammatory Responses Associated with the Induction of Hsp70 and Activation of Autophagy.

Heat stroke (HS) is a life-threatening illness and defined as when body temperature elevates above 40°C accompanied by the systemic inflammatory response syndrome that results in multiple organ dysfunctions. α-Lipoic acid (ALA) acts as a cofactor of mitochondrial enzymes and exerts anti-inflammatory and antioxidant properties in a variety of diseases. This study investigates the beneficial effects of ALA on myocardial injury and organ damage caused by experimental HS and further explores its underlying mechanism. Male Wistar rats were exposed to 42°C until their rectal core temperature reached 42.9°C and ALA was pretreared 40 or 80 mg/kg (i.v.) 1.5 h prior to heat exposure. Results showed that HS-induced lethality and hypothermia were significantly alleviated by ALA treatment that also improved plasma levels of CRE, LDH, and CPK and myocardial injury biomarkers myoglobin and troponin. In addition, ALA reduced cardiac superoxide anion formation and protein expression of cleaved caspase 3 caused by HS. Proinflammatory cytokine TNF-α and NF-κB pathways were significantly reduced by ALA treatment which may be associated with the upregulation of Hsp70. ALA significantly increased the Atg5-12 complex and LC3B II/LC3B I ratio, whereas the p62 and p-mTOR expression was attenuated in HS rats, indicating the activation of autophagy by ALA. In conclusion, ALA ameliorated the deleterious effects of HS by exerting antioxidative and anti-inflammatory capacities. Induction of Hsp70 and activation of autophagy contribute to the protective effects of ALA in HS-induced myocardial injury.

Genistein suppresses leptin-induced proliferation and migration of vascular smooth muscle cells and neointima formation.

Obesity is a strong risk factor for the development of cardiovascular diseases and is associated with a marked increase in circulating leptin concentration. Leptin is a peptide hormone mainly produced by adipose tissue and is regulated by energy level, hormones and various inflammatory mediators. Genistein is an isoflavone that exhibits diverse health-promoting effects. Here, we investigated whether genistein suppressed the atherogenic effect induced by leptin. The A10 cells were treated with leptin and/or genistein, and then the cell proliferation and migration were analysed. The reactive oxygen species (ROS) and proteins levels were also measured, such as p44/42MAPK, cell cycle-related protein (cyclin D1 and p21) and matrix metalloproteinase-2 (MMP-2). Immunohistochemistry and morphometric analysis were used for the neointima formation in a rat carotid artery injury model. Genistein (5 µM) significantly inhibited both the proliferation and migration of leptin (10 ng/ml)-stimulated A10 cells. In accordance with these finding, genistein decreased the leptin-stimulated ROS production and phosphorylation of the p44/42MAPK signal transduction pathway. Meanwhile, genistein reversed the leptin-induced expression of cyclin D1, and cyclin-dependent kinase inhibitor, p21. Genistein attenuated leptin-induced A10 cell migration by inhibiting MMP-2 activity. Furthermore, the leptin (0.25 mg/kg)-augmented neointima formation in a rat carotid artery injury model was attenuated in the genistein (5 mg/kg body weight)-treated group when compared with the balloon injury plus leptin group. Genistein was capable of suppressing the atherogenic effects of leptin in vitro and in vivo, and may be a promising candidate drug in the clinical setting.

Activation of autophagy is involved in the protective effect of 17ß-oestradiol on endotoxaemia-induced multiple organ dysfunction in ovariectomized rats.

Oestrogens have been reported to attenuate acute inflammation in sepsis. In this study, the effects of long-term oestrogen replacement with 17ß-oestradiol (E2 ) on endotoxaemia-induced circulatory dysfunction and multiple organ dysfunction syndrome were evaluated in ovariectomized (Ovx) rats. E2 (50 µg/kg, s.c., 3 times/week) was administered for 8 weeks, followed by the induction of endotoxaemia by intravenous infusion of lipopolysaccharides (LPS; 30 mg/kg/4 hrs). Oestrogen deficiency induced by ovariectomy for 9 weeks augmented the LPS-induced damage, including endotoxic shock, myocardial contractile dysfunction, renal dysfunction and rhabdomyolysis. Cardiac levels of NF-κB p65, iNOS and oxidized glutathione, free radical production in skeletal muscles, myoglobin deposition in renal tubules, and plasma levels of plasminogen activator inhibitor-1, TNF-α, and IL-6 were more pronounced in the Ovx + LPS group than in the Sham + LPS group. Long-term treatment of E2 prevented this amplified damage in Ovx rats. Six hours after LPS initiation, activation of the autophagic process, demonstrated by increases in Atg12 and LC3B-II/LC3B-I ratios, and induction of haem oxygenase (HO)-1 and heat-shock protein (HSP) 70 protein expression in myocardium were increased significantly in the Ovx + E2  + LPS group. These results suggest that activation of autophagy and induction of HO-1 and HSP70 contribute to the protective effect of long-term E2 replacement on multiple organ dysfunction syndrome in endotoxaemia.

Heat shock protein 70 and AMP-activated protein kinase contribute to 17-DMAG-dependent protection against heat stroke.

Heat shock protein 70 (Hsp70) preconditioning induces thermotolerance, and adenosine monophosphate (AMP)-activated protein kinase (AMPK) plays a role in the process of autophagy. Here, we investigated whether 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG) protected against heat stroke (HS) in rats by up-regulation of Hsp70 and phosphorylated AMPK (pAMPK). To produce HS, male Sprague-Dawley rats were placed in a chamber with an ambient temperature of 42°C. Physiological function (mean arterial pressure, heart rate and core temperature), hepatic and intestinal injury, inflammatory mediators and levels of Hsp70, pAMPK and light chain 3 (LC3B) in hepatic tissue were measured in HS rats or/and rats pre-treated with 17-DMAG. 17-DMAG pre-treatment significantly attenuated hypotension and organ dysfunction induced by HS in rats. The survival time during HS was also prolonged by 17-DMAG treatment. Hsp70 expression was increased, whereas pAMPK levels in the liver were significantly decreased in HS rats. Following pre-treatment with 17-DMAG, Hsp70 protein levels increased further, and pAMPK levels were enhanced. Treatment with an AMPK activator significantly increased the LC3BII/LC3BI ratio as a marker of autophagy in HS rats. Treatment with quercetin significantly suppressed Hsp70 and pAMPK levels and reduced the protective effects of 17-DMAG in HS rats. Both of Hsp70 and AMPK are involved in the 17-DMAG-mediated protection against HS. 17-DMAG may be a promising candidate drug in the clinical setting.

Inhibitory effects of naringenin on estrogen deficiency-induced obesity via regulation of mitochondrial dynamics and AMPK activation associated with white adipose tissue browning.

AIMS: Post-ovariectomy (OVX) changes in hormones induce obesity and white adipose tissue (WAT) inflammation. Increased energy expenditure via WAT browning is a novel therapeutic strategy for treating obesity. Naringenin (NAR) reduces inflammation and lipogenesis in obesity and attenuates estrogen deficiency-associated metabolic disorders; however, its role in WAT browning remains unclear. MATERIALS AND METHODS: We investigated NAR ability to inhibit estrogen deficiency-associated obesity in vivo using a rat model and in vitro using 3T3-L1 adipocytes. KEY FINDINGS: NAR significantly decreased the body weight and WAT mass of rats. O2 consumption, CO2 production, and energy expenditure were significantly lower in the OVX group than in the sham group, but NAR treatment reversed these effects of OVX. NAR treatment markedly improved glucose intolerance and lipid profiles as well as leptin, adiponectin, and irisin levels. NAR upregulated markers of browning and mitochondrial biogenesis in inguinal WAT. Moreover, it enhanced markers of mitochondrial fusion and inhibited fission via activating the AMP-activated protein kinase pathway. Similar results were observed in 3T3-L1 adipocytes. Moreover, NAR-induced mitochondrial biogenesis and fusion were suppressed by dorsomorphin (an AMP-activated protein kinase inhibitor). SIGNIFICANCE: NAR alleviates obesity and metabolic dysfunction through the induction of WAT browning achieved via the modulation of AMP-activated protein kinase-regulated mitochondrial dynamics in WATs. NAR supplementation may therefore represent a potential intervention for preventing postmenopausal adipose tissue dysregulation.

Alpha-Lipoic Acid Induces Adipose Tissue Browning through AMP-Activated Protein Kinase Signaling in Vivo and in Vitro.

Background: AMP-activated protein kinase (AMPK) is a key enzyme for cellular energy homeostasis and improves metabolic disorders. Brown and beige adipose tissues exert thermogenesis capacities to dissipate energy in the form of heat. Here, we investigated the beneficial effects of the antioxidant alpha-lipoic acid (ALA) in menopausal obesity and the underlying mechanisms. Methods: Female Wistar rats (8 weeks old) were subjected to bilateral ovariectomy (Ovx) and divided into four groups: Sham (n=8), Ovx (n=11), Ovx+ALA2 (n=10), and Ovx+ALA3 (n=6) (ALA 200 and 300 mg/kg/day, respectively; gavage) for 8 weeks. 3T3-L1 cells were used for in vitro study. Results: Rats receiving ALA2 and ALA3 treatment showed significantly lower levels of body weight and white adipose tissue (WAT) mass than those of the Ovx group. ALA improved plasma lipid profiles including triglycerides, total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol. Hematoxylin & eosin staining of inguinal WAT showed that ALA treatment reduced Ovx-induced adipocyte size and enhanced uncoupling protein 1 (UCP1) expression. Moreover, plasma levels of irisin were markedly increased in ALA-treated Ovx rats. Protein expression of brown fat-specific markers including UCP1, PRDM16, and CIDEA was downregulated by Ovx but markedly increased by ALA. Phosphorylation of AMPK, its downstream acetyl-CoA carboxylase, and its upstream LKB1 were all significantly increased by ALA treatment. In 3T3-L1 cells, administration of ALA (100 and 250 µM) reduced lipid accumulation and enhanced oxygen consumption and UCP1 protein expression, while inhibition of AMPK by dorsomorphin (5 µM) significantly reversed these effects. Conclusion: ALA improves estrogen deficiency-induced obesity via browning of WAT through AMPK signaling.

Inhibitory effect of ß-escin on Zika virus infection through the interruption of viral binding, replication, and stability.

ß-Escin is a mixture of triterpenoid saponins extracted from horse chestnut seeds that have diverse pharmacological activities, including anti-inflammation, anti-edematous, venotonic, and antiviral effects. In the clinical setting, ß-escin is primarily used to treat venous insufficiency and blunt trauma injuries. The anti-Zika virus (ZIKV) activity of ß-escin has not been explored. This study investigated the antiviral efficacy of ß-escin on ZIKV and dengue virus (DENV) in vitro and then elucidated the underlying mechanism. The inhibitory effects of ß-escin on viral RNA synthesis, protein levels, and infection ability were determined using qRT-PCR, Western blotting, and immunofluorescence assays, respectively. To further characterize how ß-escin interferes with the viral life cycle, the time-of-addition experiment was performed. An inactivation assay was performed to determine whether ß-escin affects ZIKV virion stability. To broaden these findings, the antiviral effects of ß-escin on different DENV serotypes were assessed using dose-inhibition and time-of-addition assays. The results showed that ß-escin exhibits anti-ZIKV activity by decreasing viral RNA levels, protein expression, progeny yield, and virion stability. ß-Escin inhibited ZIKV infection by disrupting viral binding and replication. Furthermore, ß-escin demonstrated antiviral activities against four DENV serotypes in a Vero cell model and prophylactic protection against ZIKV and DENV infections.

Ethyl pyruvate reduces acute lung injury via regulation of iNOS and HO-1 expression in endotoxemic rats.

BACKGROUND: Ethyl pyruvate (EP) has been shown to attenuate lipopolysaccharide (LPS)-induced acute lung injury (ALI). Induction of heme oxygenase-1 (HO-1) and suppression of inducible nitric oxide synthase (iNOS) expression provide cytoprotection in lung and vascular injury. The aim of this study is to evaluate whether the beneficial effect of EP on lung inflammation is related to HO-1 induction in a rat model of LPS-induced ALI. MATERIALS AND METHODS: Rats were administered LPS (30 mg/kg) by intravenous infusion for 4 h to induce ALI. EP (20, 40, and 60 mg/kg/4 h i.v. infusion) or vehicle was given 1 h after LPS initiation. RESULTS: EP 40 and 60 mg/kg attenuated plasma levels of TNF-α and IL-6 caused by LPS, and further increased IL-10 levels compared with the LPS group. At 6 h after LPS initiation, iNOS protein expression in lungs and plasma NO metabolite levels were markedly increased, which were reduced by EP 60 mg/kg. LPS caused a significant HO-1 induction, whereas administration of EP 60 mg/kg significantly induced higher HO-1 expression compared with the LPS group. The beneficial effects of EP on cytokines and iNOS expression were reversed by HO-1 inhibitor SnPP. EP significantly suppressed phosphorylated p38 MAPK and increased phosphorylated ERK1/2 protein levels in the lung tissue. The edema and infiltration of neutrophils into lungs was reduced by EP. CONCLUSION: EP reduced LPS-induced ALI, which may be mediated by induction of HO-1. The underlying mechanisms are associated with suppression of p38 MAPK and increase of ERK1/2 signaling pathway activation.

Wogonin suppresses arrhythmias, inflammatory responses, and apoptosis induced by myocardial ischemia/reperfusion in rats.

Wogonin is a flavonoid isolated from Scutellaria baicalensis Georgi, a traditional Chinese medicine, and it possesses antioxidant and anti-inflammatory effects. The aim of this study is to investigate the in vivo effect of wogonin on myocardial ischemia/reperfusion injury in an open-chest anesthetized rat model, which was induced by 45-minute left coronary artery occlusion and 2-hour reperfusion. Rats were treated with wogonin (5, 10, and 20 mg/kg, intraperitoneal) 40 minutes before ischemia or treatment with 10 mg/kg of wogonin 15 minutes after occlusion. Pretreatment with 10 mg/kg of wogonin significantly delayed the occurrence of ventricular premature contractions and tachycardia, and it suppressed the incidence of ventricular tachycardia and ventricular fibrillation, and mortality elicited by ischemia when compared with that in the control group, accompanied by reducing the arrhythmia scores. After 2-hour reperfusion, pretreatment and posttreatment with wogonin significantly reduced the infarct size and plasma levels of creatine kinase muscle-brain fraction and lactate dehydrogenase. Wogonin also significantly reduced the elevation of plasma tissue necrosis factor-α and superoxide anion production in the myocardium with ischemia/reperfusion. The expression of monocyte chemoattractant protein-1, phosphorylated p38 mitogen-activated protein kinase, p65 and IκBα, and active caspase-3 in ischemic myocardium pronouncedly increased in the control group; these were significantly attenuated by treatment with wogonin. In conclusion, wogonin demonstrated in vivo cardioprotective effects by the attenuation of the severity of ischemia-induced arrhythmias and irreversible ischemia/reperfusion injury, which is associated with its antioxidant capacity and anti-inflammatory effects. The suppression of nuclear factor-κB and p38 mitogen-activated protein kinase activation and the inhibition of monocyte chemoattractant protein-1 expression contribute to the beneficial effects of wogonin.

The Role of HSP70 in the Protective Effects of NVP-AUY922 on Multiple Organ Dysfunction Syndrome in Endotoxemic Rats.

Sepsis is defined as a life-threatening organ dysfunction syndrome with high morbidity and mortality caused by bacterial infection. The major characteristics of sepsis are systemic inflammatory responses accompanied with elevated oxidative stress, leading to multiple organ dysfunction syndrome (MODS), and disseminated intravascular coagulation (DIC). As a molecular chaperon to repair unfolded proteins, heat shock protein 70 (HSP70) maintains cellular homeostasis and shows protective effects on inflammatory damage. HSP 90 inhibitors were reported to exert anti-inflammatory effects via activation of the heat shock factor-1 (HSF-1), leading to induction of HSP70. We evaluated the beneficial effect of HSP 90 inhibitor NVP-AUY 922 (NVP) on multiple organ dysfunction syndrome induced by lipopolysaccharide (LPS) and further explored the underlying mechanism. NVP (5 mg/kg, i.p.) was administered 20 h prior to LPS initiation (LPS 30 mg/kg, i.v. infusion for 4 h) in male Wistar rats. Results demonstrated that pretreatment with NVP significantly increased survival rate and prevented hypotension at 6 h after LPS injection. Plasma levels of ALT, CRE and LDH as well as IL-1ß and TNF-α were significantly reduced by NVP at 6 h after LPS challenge. The induction of inducible NO synthase in the liver, lung and heart and NF-κB p-p65 and caspase 3 protein expression in the heart were also attenuated by NVP. In addition, NVP markedly induced HSP70 and HO-1 proteins in the liver, lung and heart after LPS injection. These results indicated that NVP possessed the anti-inflammatory and antioxidant effects on LPS-induced acute inflammation, which might be associated with HSP70 and HO-1, leading to prevent MODS in sepsis. NVP might be considered as a novel therapeutic strategy in the prevention of sepsis-induced MODS.

Involvement of HO-1 and Autophagy in the Protective Effect of Magnolol in Hepatic Steatosis-Induced NLRP3 Inflammasome Activation In Vivo and In Vitro.

Magnolol (MG) is the main active compound of Magnolia officinalis and exerts a wide range of biological activities. In this study, we investigated the effects of MG using tyloxapol (Tylo)-induced (200 mg/kg, i.p.) hyperlipidemia in rats and palmitic acid (PA)-stimulated (0.3 mM) HepG2 cells. Our results showed that Tylo injection significantly increased plasma levels of triglyceride and cholesterol as well as superoxide anion in the livers, whereas MG pretreatment reversed these changes. MG reduced hepatic lipogenesis by attenuating sterol regulatory element-binding protein-1c (SREBP-1c) and fatty acid synthase (FAS) proteins and Srebp-1, Fas, Acc, and Cd36 mRNA expression as well as upregulated the lipolysis-associated genes Hsl, Mgl, and Atgl. Furthermore, MG reduced plasma interleukin-1ß (IL-1ß) and protein expression of NLR family pyrin domain-containing 3 (NLRP3), apoptosis-associated speck-like protein (ASC), and caspase 1 as well as upregulated nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and induction of heme oxygenase-1 (HO-1) in hepatocytes of Tylo-treated rats. Enhanced autophagic flux by elevation of autophagy related protein 5-12 (ATG5-12), ATG7, Beclin1, and microtubule-associated protein light chain 3 B II (LC3BII)/LC3BI ratio, and reduction of sequestosome-1 (SQSTM1/p62) and phosphorylation of mTOR was observed by MG administration. However, autophagy inhibition with 3-methyladenine (3-MA) in HepG2 cells drastically abrogated the MG-mediated suppression of inflammation and lipid metabolism. In conclusion, MG inhibited hepatic steatosis-induced NLRP3 inflammasome activation through the restoration of autophagy to promote HO-1 signaling capable of ameliorating oxidative stress and inflammatory responses.

Involvement of the p62/Nrf2/HO-1 pathway in the browning effect of irisin in 3T3-L1 adipocytes.

Irisin has gained attention because of its potential applications in the treatment of metabolic diseases. Accumulating evidence indicates that irisin attenuates obesity via the browning of white adipose tissue; however, the underlying mechanisms are unclear. Here, we evaluated the effects of irisin on adipocyte browning and the underlying mechanisms. The western blotting and immunofluorescence analyses demonstrated that irisin significantly induced the up-regulation of brown fat-specific proteins (PGC1α, PRDM16, and UCP-1) and HO-1 in 3T3-L1 adipocytes. Moreover, irisin significantly increased the levels of cytosolic p62 and nuclear Nrf2. These effects of irisin in the adipocytes were attenuated by treatment with SnPP or p62 siRNA. In addition, the browning effect of irisin was observed in BAT-WT-1 cells. These findings suggest that irisin induced browning effect via the p62/Nrf2/HO-1 signalling pathway and that it may be a potential candidate for preventing or treating obesity.

17-DMAG, an Hsp90 inhibitor, ameliorates ovariectomy-induced obesity in rats.

AIMS: Obesity is not only associated with metabolic diseases but is also a symptom of menopause in women. To date, there are no effective drugs for the management of obesity, and it is important to find new agents with fewer side effects, for the treatment of obesity. This study aimed to determine the anti-obesity effect of 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), a heat shock protein 90 (Hsp90) inhibitor, and its underlying mechanism in rats with ovariectomy-induced obesity. MAIN METHODS: Ovariectomy (Ovx) rats were treated with 17-DMAG (1 mg kg-1, intraperitoneally) for eight weeks from one week after surgery. The body weight, food intake, locomotor activity, adipogenic- and autophagy-related protein expression in white adipose tissue (WAT) and plasma triglyceride (TG) levels were measured in sham and Ovx rats. KEY FINDINGS: Compared with sham rats, Ovx rats showed increased weight gain, food intake, WAT mass, TG levels, adipogenic protein expression, and decreased locomotor activity. Furthermore, autophagy-related proteins and Foxo3a of WAT were significantly increased in Ovx rats. However, with the exclusion of increased food intake, the changes induced by Ovx were all reversed in 17-DMAG-treated Ovx rats. In addition, the expression of Hsp70 and phosphorylation of Akt increased in 17-DMAG-treated Ovx rats. SIGNIFICANCE: These results suggest that 17-DMAG significantly ameliorated obesity induced by Ovx, and this phenomenon is accompanied by the downregulation of adipogenic-related and autophagy-related proteins as well as the upregulation of Akt-phosphorylation and Hsp70 expression. Therefore, 17-DMAG may be a potential agent for preventing or treating obesity in postmenopausal women.

Genistein ameliorated obesity accompanied with adipose tissue browning and attenuation of hepatic lipogenesis in ovariectomized rats with high-fat diet.

Estrogen deficiency in postmenopausal women is linked to the higher prevalence of obesity, type 2 diabetes and metabolic syndromes. Development of beige adipocytes (browning of WAT) increases energy expenditure and could be a promising strategy for obesity management. This study aimed to investigate the effects of phytoestrogen genistein (GEN) on white adipose tissue (WAT) inflammation, browning and hepatic lipogenesis in ovariectomized rats with high-fat diet (HFD) and further explore the underlying mechanism. Female Wistar rats received ovariectomy (Ovx) and HFD (45% fat) and then were administered with 17ß-estradiol (E2, 3 times/week, subcutaneously) or GEN (15 mg/kg or 30 mg/kg, gavage, once daily) for 4 weeks. Administration of GEN decreased Ovx-induced body weight gain and adiposity and improved insulin sensitivity as well as increased insulin signaling p-IRS1 and p-AKT in retroperitoneal WAT. Adipocyte hypertrophy and production of proinflammatory cytokines MCP-1, TNF-α and IL-6 were reduced by GEN. It also suppressed the activation of NF-κB pathway evidenced by attenuation of p65 and phospho-IκB levels. Additionally, GEN elevated myokine irisin and promoted WAT browning by increasing UCP-1, PRDM-16, PGC-1α and CIDEA proteins and Ppargc1a, Ucp-1 and Tbx-1 mRNA in inguinal WAT which is associated with up-regulation of nuclear estrogen receptor-α. Plasma levels of triglyceride and cholesterol were reduced by GEN treatment accompanied with inhibition of lipogenic proteins (p-ACC, SREBP-1, FAS and CD36) in the liver. Long-term treatment with GEN attenuated estrogen-deficiency-induced obesity, WAT inflammation and hepatic lipogenesis and promoted the induction of WAT browning. It may provide a promising approach to prevent obesity during menopause.

Anti-inflammatory effects of LK-3, on LPS-induced sepsis in rats.

Dextromethorphan (DM), an antitussive agent, has been shown to have anti-inflammatory and immunomodulatory effects in vitro. Thus, the aim of this study was to evaluate the effects of LK-3, an analog of DM, on sepsis induced by intravenous (i.v.) administration of lipopolysaccharide (LPS; 10 mg/ kg) in anesthetized Wistar rats. Results demonstrated that post-treatment with LK-3 (4 mg/kg, i.v.) significantly attenuated the deleterious hemodynamic changes (e.g., hypotension and bradycardia) in rats treated with LPS. Meanwhile, LK-3 (4 mg/kg) significantly inhibited the elevation of plasma tumor necrosis factor-alpha, as well as values of glutamate-oxalacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) caused by LPS. The induction of inducible NO synthase and the overproduction of NO and superoxide anions by LPS were also reduced by post-treatment of LK-3. Moreover, infiltration of neutrophils into the lungs and liver of rats 8 h after treatment with LPS was also reduced by post-treatment with LK-3. In conclusion, the beneficial effects of LK-3 on LPS-induced sepsis resulted from its anti-inflammatory and antioxidant effects.

Raspberry ketone induces brown-like adipocyte formation through suppression of autophagy in adipocytes and adipose tissue.

Promoting white adipose tissue (WAT) to acquire brown-like characteristics is a promising approach for obesity treatment. Although raspberry ketone (RK) has been reported to possess antiobesity activity, its effects on the formation of brown-like adipocytes remain unclear. Therefore, we investigated the effects and underlying mechanism of RK on WAT browning in 3T3-L1 adipocytes and rats with ovariectomy (Ovx)-induced obesity. RK (100 µM) significantly induced browning of 3T3-L1 cells by increasing mitochondrial biogenesis and the expression of browning-specific proteins (PR domain containing 16, PRDM16; peroxisome proliferator-activated receptor gamma coactivator 1-alpha, PGC-1α; uncoupling protein-1, UCP-1) and lipolytic enzymes (hormone-sensitive lipase and adipose triglyceride lipase). RK significantly reduced the expression of the autophagy-related protein Atg12 and increased the expression of p62 and heme oxygenase 1 (HO-1). Additionally, these effects of RK were reversed by the HO-1 inhibitor SnPP (20 µM). In addition, RK (160 mg/kg, gavage, for 8 weeks) significantly reduced body weight gain (Ovx+RK, 191.8 ± 4.6 g vs. Ovx, 223.6 ± 5.9; P < .05), food intake, the amount of inguinal adipose tissue (Ovx+RK, 9.05 ± 1.1 g vs Ovx, 12.9 ± 0.92 g; P < .05) and the size of white adipocytes in Ovx rats. Moreover, compared to expression in the Ovx group, the levels of browning-specific proteins were significantly higher and the levels of autophagy-related proteins were significantly lower in the Ovx+RK group. Therefore, this study elucidated the mechanism associated with RK-induced WAT browning and thus provides evidence to support the clinical use of RK for obesity treatment.

The involvement of AMP-activated protein kinases in the anti-inflammatory effect of nicotine in vivo and in vitro.

AMP-activated protein kinase (AMPK) is the downstream component of a kinase cascade that plays a pivotal role in energy homeostasis. AMPK has recently emerged as an attractive and novel target for inflammatory disorders. Thus, the aim of this study was to assess the role of AMPKalpha in the anti-inflammatory effect of nicotine in carrageenan-induced rat paw edema model and to evaluate the mechanism of nicotine-induced AMPKalpha phosphorylation in RAW 264.7 cells. The results indicate that nicotine alleviated paw edema and the activation of AMPKalpha involved in the anti-inflammatory effect of nicotine in vivo. In addition, nicotine was able to activate AMPKalpha phosphorylation in macrophages and this effect was mediated through nicotinic acetylcholine receptors. Furthermore, nicotine significantly induced the phosphorylation of Akt and the Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) protein expression in macrophages. Wortmannin, a specific inhibitor of phosphotidylinositol 3-kinase (PI3K), suppressed nicotine-induced Akt and AMPKalpha phosphorylation. STO-609, a CaMKK inhibitor, not only inhibited the activation of AMPKalpha but also suppressed the phosphorylation of Akt induced by nicotine. In conclusion, both of CaMKK and PI3K/Akt pathways are involved in the nicotine-induced AMPKalpha phosphorylation in macrophages, and the interaction of CaMKK and Akt may exist. AMPKalpha is a novel and critical component of anti-inflammatory effect of nicotine.