High Uric Acid Orchestrates Ferroptosis to Promote Cardiomyopathy Via ROS-GPX4 Signaling.

Aims: High uric acid (HUA), as a pro-oxidant, plays a significant role in the pathophysiology of cardiovascular disease. Studies have indicated that elevated uric acid levels can adversely affect cardiovascular health. Nevertheless, the impact of hyperuricemia on cardiomyopathy remains uncertain. Further research is needed to elucidate the relationship between HUA and cardiomyopathy, shedding light on its potential implications for heart health. Results: We demonstrated that uricase knockout (Uox-KO) mice accelerated the development of cardiomyopathy, causing significantly impaired cardiac function and myocardial fibrosis. Meanwhile, the mitochondrial morphology was destroyed, the lipid peroxidation products increased in number and the antioxidant function was weakened. In addition, we evaluated the effects of ferrostatin-1 (Fer-1), the ferroptosis inhibitor. Myocardial damage can be reversed by the Fer-1 treatment caused by HUA combined with doxorubicin (DOX) treatment. Benzbromarone, a uric acid-lowering drug, decreases myocardial fibrosis, and ferroptosis by alleviating hyperuricemia in Uox-KO mice by DOX administration. In vitro, we observed that the activity of cardiomyocytes treated with HUA combined with DOX decreased significantly, and lipid reactive oxygen species (ROS) increased significantly. Afterward, we demonstrated that HUA can promote oxidative stress in DOX, characterized by increased mitochondrial ROS, and downregulate protein levels of glutathione peroxidase 4 (GPX4). N-acetyl-l-cysteine, an antioxidant, inhibits the process by which HUA promotes DOX-induced ferroptosis by increasing the GPX4 expression. Innovation: We verified that HUA can exacerbate myocardial damage. This has clinical implications for the treatment of cardiac damage in patients with hyperuricemia. Conclusions: Our data suggested that HUA promotes the cardiomyopathy. HUA promotes DOX-induced ferroptosis by increasing oxidative stress and downregulating GPX4. Antioxid. Redox Signal. 00, 00-00.

RAGE in circulating immune cells is fundamental for hippocampal inflammation and cognitive decline in a mouse model of latent chronic inflammation.

BACKGROUND: Latent chronic inflammation has been proposed as a key mediator of multiple derangements in metabolic syndrome (MetS), which are increasingly becoming recognized as risk factors for age-related cognitive decline. However, the question remains whether latent chronic inflammation indeed induces brain inflammation and cognitive decline. METHODS: A mouse model of latent chronic inflammation was constructed by a chronic subcutaneous infusion of low dose lipopolysaccharide (LPS) for four weeks. A receptor for advanced glycation end products (RAGE) knockout mouse, a chimeric myeloid cell specific RAGE-deficient mouse established by bone marrow transplantation and a human endogenous secretory RAGE (esRAGE) overexpressing adenovirus system were utilized to examine the role of RAGE in vivo. The cognitive function was examined by a Y-maze test, and the expression level of genes was determined by quantitative RT-PCR, western blot, immunohistochemical staining, or ELISA assays. RESULTS: Latent chronic inflammation induced MetS features in C57BL/6J mice, which were associated with cognitive decline and brain inflammation characterized by microgliosis, monocyte infiltration and endothelial inflammation, without significant changes in circulating cytokines including TNF-α and IL-1ß. These changes as well as cognitive impairment were rescued in RAGE knockout mice or chimeric mice lacking RAGE in bone marrow cells. P-selectin glycoprotein ligand-1 (PSGL-1), a critical adhesion molecule, was induced in circulating mononuclear cells in latent chronic inflammation in wild-type but not RAGE knockout mice. These inflammatory changes and cognitive decline induced in the wild-type mice were ameliorated by an adenoviral increase in circulating esRAGE. Meanwhile, chimeric RAGE knockout mice possessing RAGE in myeloid cells were still resistant to cognitive decline and brain inflammation. CONCLUSIONS: These findings indicate that RAGE in inflammatory cells is necessary to mediate stimuli of latent chronic inflammation that cause brain inflammation and cognitive decline, potentially by orchestrating monocyte activation via regulation of PSGL-1 expression. Our results also suggest esRAGE-mediated inflammatory regulation as a potential therapeutic option for cognitive dysfunction in MetS with latent chronic inflammation.

AMPD2 plays important roles in regulating hepatic glucose and lipid metabolism.

Dysregulation of hepatic glucose and lipid metabolism can instigate the onset of various metabolic disorders including obesity, dyslipidemia, insulin resistance, type 2 diabetes, and fatty liver disease. Adenosine monophosphate (AMP) deaminase (AMPD), which converts AMP to inosine monophosphate, plays a key role in maintaining adenylate energy charge. AMPD2 is the major isoform present in the liver. However, the mechanistic link between AMPD2 and hepatic glucose and lipid metabolism remains elusive. In this study, we probed into the hepatic glucose and lipid metabolism in AMPD2-deficient (A2-/-) mice. These mice exhibited reduced body weight, fat accumulation, and blood glucose levels, coupled with enhanced insulin sensitivity while maintaining consistent calorie intake and spontaneous motor activity compared with wild type mice. Furthermore, A2-/- mice showed mitigated obesity and hyper-insulinemia induced by high-fat diet (HFD) but elevated levels of the serum triglyceride and cholesterol. The hepatic mRNA levels of several fatty acid and cholesterol metabolism-related genes were altered in A2-/- mice. RNA sequencing unveiled multiple alterations in lipid metabolic pathways due to AMPD2 deficiency. These mice were also more susceptible to fasting or HFD-induced hepatic lipid accumulation. The liver exhibited elevated AMP levels but unaltered AMP/ATP ratio. In addition, AMPD2 deficiency is not associated with the adenosine production. In summary, this study established a link between purine metabolism and hepatic glucose and lipid metabolism via AMPD2, providing novel insights into these metabolic pathways.

Activation of p62-NRF2 Axis Protects against Doxorubicin-Induced Ferroptosis in Cardiomyocytes: A Novel Role and Molecular Mechanism of Resveratrol.

Doxorubicin (DOX) is a most common anthracycline chemotherapeutic agent; however, its clinical efficacy is limited due to its severe and irreversible cardiotoxicity. Ferroptosis, characterized by iron overload and lipid peroxidation, plays a pivotal role in DOX-induced cardiotoxicity. Resveratrol (RSV) displays cardioprotective and anticancer effects, owing to its antioxidative and anti-inflammatory properties. However, the role and mechanism of RSV in DOX-mediated ferroptosis in cardiomyocytes is unclear. This study showed that DOX decreased cell viability, increased iron accumulation and lipid peroxidation in H9c2 cells; however, these effects were reversed by RSV and ferroptosis inhibitor ferrostatin-1 (Fer-1) pre-treatment. Additionally, RSV significantly increased the cell viability of H9c2 cells treated with ferroptosis inducers Erastin (Era) and RSL3. Mechanistically, RSV inhibited mitochondrial reactive oxygen species (mtROS) overproduction and upregulated the p62-NRF2/HO-1 pathway. RSV-induced NRF2 activation was partially dependent on p62, and the selective inhibition of p62 (using p62-siRNA interference) or NRF2 (using NRF2 specific inhibitor, ML385) significantly abolished the anti-ferroptosis function of RSV. Furthermore, RSV treatment protected mice against DOX-induced cardiotoxicity, including significantly improving left ventricular function, ameliorating myocardial fibrosis and suppressing ferroptosis. Consistent with in vitro results, RSV also upregulated the p62-NRF2/HO-1 expression, which was inhibited by DOX, in the myocardium. Notably, the protective effect of RSV in DOX-mediated ferroptosis was similar to that of Fer-1 in vitro and in vivo. Thus, the p62-NRF2 axis plays a critical role in regulating DOX-induced ferroptosis in cardiomyocytes. RSV as a potent p62 activator has potential as a therapeutic target in preventing DOX-induced cardiotoxicity via ferroptosis modulation.

Hyperuricemia contributes to glucose intolerance of hepatic inflammatory macrophages and impairs the insulin signaling pathway via IRS2-proteasome degradation.

Aim: Numerous reports have demonstrated the key importance of macrophage-elicited metabolic inflammation in insulin resistance (IR). Our previous studies confirmed that hyperuricemia or high uric acid (HUA) treatment induced an IR state in several peripheral tissues to promote the development of type 2 diabetes mellitus (T2DM). However, the effect of HUA on glucose uptake and the insulin sensitivity of macrophages and its mechanism is unclear. Methods: To assess systemic IR, we generated hyperuricemic mice by urate oxidase knockout (UOX-KO). Then, glucose/insulin tolerance, the tissue uptake of 18F-fluorodeoxyglucose, body composition, and energy balance were assessed. Glucose uptake of circulating infiltrated macrophages in the liver was evaluated by glucose transporter type 4 (GLUT-4) staining. Insulin sensitivity and the insulin signaling pathway of macrophages were demonstrated using the 2-NBDG kit, immunoblotting, and immunofluorescence assays. The immunoprecipitation assay and LC-MS analysis were used to determine insulin receptor substrate 2 (IRS2) levels and its interacting protein enrichment under HUA conditions. Results: Compared to WT mice (10 weeks old), serum uric acid levels were higher in UOX-KO mice (WT, 182.3 ± 5.091 µM versus KO, 421.9 ± 45.47 µM). Hyperuricemic mice with metabolic disorders and systemic IR showed inflammatory macrophage recruitment and increased levels of circulating proinflammatory cytokines. HUA inhibited the nuclear translocation of GLUT-4 in hepatic macrophages, restrained insulin-induced glucose uptake and glucose tolerance, and blocked insulin IRS2/PI3K/AKT signaling. Meanwhile, HUA mediated the IRS2 protein degradation pathway and activated AMPK/mTOR in macrophages. LC-MS analysis showed that ubiquitination degradation could be involved in IRS2 and its interacting proteins to contribute to IR under HUA conditions. Conclusion: The data suggest that HUA-induced glucose intolerance in hepatic macrophages contributed to insulin resistance and impaired the insulin signaling pathway via IRS2-proteasome degradation.

Receptor of Advanced Glycation End Products Deficiency Attenuates Cisplatin-Induced Acute Nephrotoxicity by Inhibiting Apoptosis, Inflammation and Restoring Fatty Acid Oxidation.

Cisplatin is a widely used and potent anti-neoplastic agent, but severe and inescapable side effects in multiple normal tissues and organs limit its application, especially nephrotoxicity. Molecular mechanisms of cisplatin nephrotoxicity involve mitochondrial damage, oxidative stress, endoplasmic reticulum stress, inflammation, apoptosis, necroptosis, etc. Receptor of advanced glycation end products (RAGE) is a multiligand pattern recognition receptor, engaged in inflammatory signaling and mitochondrial homeostasis. Whether inhibition of RAGE alleviates cisplatin-induced nephropathy has not been investigated. Here, we revealed that RAGE deficiency attenuates cisplatin-induced acute nephrotoxicity, as evidenced by reduced apoptosis, inflammation, lipid accumulation, restored mitochondrial homeostasis and fatty acid oxidation in renal tubular epithelial cells (TECs). In vitro studies showed that, the RAGE-specific inhibitor FPS-ZM1 attenuated the cisplatin-induced decrease of cell viability and fatty acid oxidation in the normal rat renal TEC line NRK-52E cells. Taken together, RAGE knockout mitigated cisplatin-induced acute nephrotoxicity by inhibiting apoptosis, inflammation, and restoring fatty acid oxidation in TECs, suggesting that RAGE inhibition could be a therapeutic option for cisplatin-induced acute nephrotoxicity.

Autophagy protects against high uric acid-induced hepatic insulin resistance.

Uric acid (UA), the end-product of purine metabolism, is closely related to hepatic insulin resistance (IR). Autophagy is a conserved intracellular degradation process maintaining cellular homeostasis. Autophagy plays a protective role in obesity-related hepatic IR, but whether it occurs in high uric acid (HUA)-induced hepatic IR is unclear. In this study, spontaneously elevated UA level induced hepatic IR and facilitated hepatic autophagy degradation in uricase knockout (Uox-/-) mice. In vitro, HepG2 cells stimulated with HUA medium showed decreased glucose uptake and inhibition of insulin signaling pathways, concomitant with activation of autophagy, as manifested by increased conversion of LC3B-I to -II. Rapamycin, the autophagy activator, alleviated but the autophagy inhibitor trimethyl adenine (3-MA) aggravated HUA-induced IR in HepG2 cells. Similarly, rapamycin ameliorated and 3-MA worsened HUA-induced blood glucose level and hepatic IR in Uox-/- mice. Mechanistically, HUA enhanced AMPKα phosphorylation (p-AMPKα) and inhibited mammalian target of rapamycin phosphorylation (p-mTOR) in HepG2 cells. The levels of p-AMPKα and LC3B-II/I were downregulated in HepG2 cells transfected with small interfering RNA (siRNA) against AMPKα, which suggests that the AMPKα-mTOR pathway was involved in HUA-induced autophagy. Antioxidant N-acetyl-L-cysteine reversed elevated reactive oxygen species levels induced by HUA in HepG2 cells, and AMPKα level was also inhibited, which suggests that AMPKα activation may be derived from reactive oxygen species. Collectively, these findings demonstrate that HUA increased hepatic autophagy, and autophagy activation plays a protective role in hepatic IR, which may suggest a potential therapeutic target for hepatic IR derived from HUA.

Silencing TXNIP ameliorates high uric acid-induced insulin resistance via the IRS2/AKT and Nrf2/HO-1 pathways in macrophages.

Insulin resistance (IR) promotes atherosclerosis and increases the risk of diabetes and cardiovascular diseases. Our previous studies have demonstrated that high uric acid (HUA) increased oxidative stress, leading to IR in cardiomyocytes and pancreatic ß cells. However, whether HUA can induce IR in monocytes/macrophages, which play critical roles in all stages of atherosclerosis, is unclear. Recent findings revealed that thioredoxin-interacting protein (TXNIP) negatively regulates insulin signaling; however, the roles and mechanisms of TXNIP in HUA-induced IR remain unclear. Therefore, in this study, we investigated the function of TXNIP in macrophages treated with UA. Transcriptomic profiling revealed TXNIP as one of the most upregulated genes, and subsequent RT-PCR and Western blot analyses confirmed that TXNIP was upregulated by HUA. HUA treatment significantly increased mitochondrial reactive oxygen species (MtROS) levels and decreased insulin-stimulated glucose uptake. Silencing TXNIP by RNA interference significantly diminished HUA-induced oxidative stress and IR. Mechanistically, silencing TXNIP reversed the inhibition of the phosphorylation of insulin receptor substrate 2 (IRS2)/protein kinase B (AKT) pathway induced by HUA. Additional study revealed that HUA induced the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) signaling pathway, but silencing TXNIP abolished it. Moreover, Nrf2 inhibitor (ML385) ameliorated HUA-induced IR independent of IRS2/AKT signaling. Probenecid, a well-known UA-lowering drug, significantly suppressed the activation of TXNIP and Nrf2/HO-1 signaling. Furthermore, RNA-seq revealed that activation of the TXNIP-related redox pathway may be a key regulator in patients with asymptomatic hyperuricemia. These data suggest that silencing TXNIP could ameliorate HUA-induced IR via the IRS2/AKT and Nrf2/HO-1 pathways in macrophages. Additionally, TXNIP might be a promising therapeutic target for preventing and treating oxidative stress and IR induced by HUA.

Underweight and obesity are strong predictors of clinical outcomes in patients with ankylosing spondylitis: data from the Smart-phone SpondyloArthritis Management System.

BACKGROUND: The aim of this study was to examine the impact of underweight, overweight and obesity on clinical outcomes and treatment responses to biologics in Chinese patients with ankylosing spondylitis (AS). METHODS: Body mass index (BMI) was available in 1074 patients from the Smart-phone SpondyloArthritis Management System. Patients were categorized into four groups based on BMI: underweight, normal weight, overweight and obesity. Multivariable median regression analyses examined the effect of underweight and obesity on clinical outcomes and treatment response to biologics. RESULTS: Among 1074 patients with AS, normal weight accounted for 49.1%, while underweight, overweight, and obesity for 8.1%, 30.1%, and 12.0%, respectively. Compared to patients with normal weight, patients with underweight, overweight and obesity had an increased disease activity, while patients with underweight and obesity had a significantly poor Bath Ankylosing Spondylitis Functional Index and Assessment of Spondyloarthritis International Society Health Index scores. For tumor necrosis factor (TNF)-α inhibitor users, BMI was found to be negatively correlated with changes in disease activity in the multivariate regression model (all p < 0.05). Besides, the patients using TNF-α inhibitor in the overweight or obesity categories were much less likely to achieve a significant reduction on disease activity during follow-up period in the multivariate regression model (all p < 0.05), taking these with normal-weight patients as a reference. CONCLUSIONS: Both underweight and obesity except for overweight were associated independently with worse disease activity, physical function and health status. Overweight and obesity might impact on treatment responses to biologics in patients with AS. This argues that weight management, to maintain it at a normal level, should be one of the disease management strategies in patients with AS.

High uric acid induces liver fat accumulation via ROS/JNK/AP-1 signaling.

Uric acid is the end metabolite derived from the oxidation of purine compounds. Overwhelming evidence shows the vital interrelationship between hyperuricemia (HUA) and nonalcoholic fatty liver disease (NAFLD). However, the mechanisms for this association remain unclear. In this study, we established a urate oxidase-knockout (Uox-KO) mouse model by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology. To study the correlation between HUA and NAFLD, human HepG2 hepatoma cells were treated in culture medium with high level of uric acid. In vivo, the Uox-KO mice spontaneously developed hyperuricemia and aberrant lipid-metabolism, concomitant with abnormal hepatic fat accumulation. HUA activated c-Jun N-terminal kinase (JNK) in vivo and in vitro. Furthermore, inhibiting JNK activation by a JNK-specific inhibitor, SP600125, decreased fat accumulation and lipogenic gene expression induced by HUA. Overexpression of the lipogenic enzymes fatty acid synthase and acetyl-CoA carboxylase 1 was via activation of JNK, which was blocked by the JNK inhibitor SP600125. HUA activated AP-1 to upregulate lipogenic gene expression via JNK activation. In addition, HUA caused mitochondrial dysfunction and reactive oxygen species production. Pretreatment with the antioxidant N-acetyl-l-cysteine could ameliorate HUA-activated JNK and hepatic steatosis. These data suggest that ROS/JNK/AP-1 signaling plays an important role in HUA-mediated fat accumulation in liver.NEW & NOTEWORTHY Hyperuricemia and nonalcoholic fatty liver disease are global public health problems, which are strongly associated with metabolic syndrome. In this study, we demonstrate that uric acid induces hepatic fat accumulation via the ROS/JNK/AP-1 pathway. This study identifies a new mechanism of NAFLD pathogenesis and new potential therapeutic strategies for HUA-induced NAFLD.

Uric acid inhibits HMGB1-TLR4-NF-κB signaling to alleviate oxygen-glucose deprivation/reoxygenation injury of microglia.

Mounting evidence has implicated inflammation in ischemia-reperfusion injury following acute ischemic stroke (AIS). Microglia remain the primary initiator and participant of brain inflammation. Emerging evidence has indicated that uric acid has promise for the treatment of AIS, but its explicit mechanisms remain elusive. Here, we observed that uric acid reduced the severity of cerebral infarction and attenuated the activation of microglia in the cerebral cortex in a mouse middle cerebral-artery occlusion/reperfusion model. Thus, we speculated that uric acid may play a role by directly interfering with the inflammatory response of microglia. First, we investigated whether the HMGB1-TLR4-NF-κB signaling plays a role in oxygen glucose deprivation and reperfusion (OGD/R) injury of BV2 cells. Inhibition of the signaling significantly reduced the release of the proinflammatory cytokines tumor necrosis factor α (TNF-α), interleukin 1ß (IL1ß), and IL6 caused by OGD/R in BV2 cells. Second, uric acid weakened the decreased cell viability and lactate dehydrogenase release induced by OGD/R in BV2 cells. Finally, uric acid reduced the release of the proinflammatory cytokines TNF-α, IL1ß, and IL6 caused by OGD/R in BV2 cells by dampening HMGB1-TLR4-NF-κB signaling, which was reversed by probenecid treatment, an inhibitor of the uric acid channel. Hence, uric acid halted the release of inflammatory factors and the decreased cell viability induced by ODG/R via inhibiting the microglia HMGB1-TLR4-NF-κB signaling, thereby alleviating the damage to microglia. This may be part of the molecular mechanisms by which uric acid protects mice against the brain damage of middle cerebral-artery occlusion/reperfusion.

High uric acid promotes dysfunction in pancreatic ß cells by blocking IRS2/AKT signalling.

Hyperuricaemia is a disorder of purine metabolism. Elevated serum uric acid is strongly associated with many diseases, including gout, abdominal obesity, insulin resistance, and cardiovascular and kidney disease. Our previous studies showed that high uric acid (HUA) induced insulin resistance in several peripheral organs, including the liver, myocardium and adipose tissue. However, whether HUA directly induces insulin resistance of pancreatic ß cells, the only source of insulin in the body and also a sensitive insulin target, is unknown. In this study, pancreatic ß cells pretreated with HUA showed impaired insulin expression/secretion, glucose uptake and the glycolytic pathway. RNA-seq revealed that HUA affected the biological processes of INS-1 cells broadly, including oxidoreduction coenzyme metabolic process, pyruvate metabolic process, and glycolytic process. In addition, HUA reduced mitochondrial membrane potential and increased the production of reactive oxygen species(ROS) in INS-1 cells. INS-1 cells pretreated with probenecid, an organic anion transporter inhibitor, protected INS-1 cells against HUA-induced insulin secretion decrease, Pretreatment with N-acetyl-L-cysteine(NAC), a globally used antioxidant, recovered HUA-decreased insulin secretion and glucose uptake by pancreatic ß cells. Insulin-like growth factor 1 (IGF-1), the phosphatidylinositol 3-kinase (PI3K) activator, rescues HUA-decreased insulin secretion by re-activating AKT phosphorylation. Thus, HUA induce insulin resistance, impaired insulin secretion and glycolytic pathway of pancreatic ꞵ cell through IRS2/AKT pathway.

The role and molecular mechanism of FoxO1 in mediating cardiac hypertrophy.

Cardiac hypertrophy can lead to heart failure and cardiovascular events and has become a research hotspot in the field of cardiovascular disease. Despite extensive and in-depth research, the pathogenesis of cardiac hypertrophy is far from being fully understood. Increasing evidence has shown that the transcription factor forkhead box protein O 1 (FoxO1) is closely related to the occurrence and development of cardiac hypertrophy. This review summarizes the current literature on the role and molecular mechanism of FoxO1 in cardiac hypertrophy. We searched the database MEDLINE via PubMed for available evidence on the effect of FoxO1 on cardiac hypertrophy. FoxO1 has many effects on multiple diseases, including cardiovascular diseases, diabetes, cancer, aging, and stem cell activity. Recent studies have shown that FoxO1 plays a critical role in the development of cardiac hypertrophy. Evidence for this relationship includes the following. (i) FoxO1 can regulate cardiac growth/protein synthesis, calcium homeostasis, cell apoptosis, and autophagy and (ii) is controlled by several upstream signalling molecules (e.g. phosphatidylinositol 3-kinase/Akt, AMP-activated protein kinase, and sirtuins) and regulates many downstream transcription proteins (e.g. ubiquitin ligases muscle RING finger 1/muscle atrophy F-box, calcineurin/nuclear factor of activated T cells, and microRNAs). In response to stress or external stimulation (e.g. low energy, oxidative stress, or growth factor signalling), FoxO1 undergoes post-translational modification and transfers from the cytoplasm to nucleus, thus regulating the expression of a series of target genes in myocardium that are involved in cardiac growth/protein synthesis, calcium homeostasis, cell apoptosis, and autophagy. (iii) Finally, targeted regulation of FoxO1 is an effective method of intervening in myocardial hypertrophy. The information reviewed here should be significant for understanding the roles of FoxO1 in cardiac hypertrophy and should contribute to the design of further studies related to FoxO1 and the hypertrophic response. It should also shed light on a potential treatment for cardiac hypertrophy.

High Uric Acid Inhibits Cardiomyocyte Viability Through the ERK/P38 Pathway via Oxidative Stress.

BACKGROUND/AIMS: Clinical studies have shown that hyperuricaemia is strongly associated with cardiovascular disease. However, the molecular mechanisms of high uric acid (HUA) associated with cardiovascular disease remain poorly understood. In this study, we investigated the effect of HUA on cardiomyocytes. METHODS: We exposed H9c2 cardiomyocytes to HUA, then cell viability was determined by MTT assay, and reactive oxygen species' (ROS) production was detected by a fluorescence assay. Western blot analysis was used to examine phosphorylation of extracellular signal-regulated kinase (ERK), p38, phosphatidylinositol 3-kinase (PI3K) and Akt. We monitored the impact of HUA on phospho-ERK and phospho-p38 levels in myocardial tissue from an acute hyperuricaemia mouse model established by potassium oxonate treatment. RESULTS: HUA decreased cardiomyocyte viability and increased ROS production in cardiomyocytes; pre-treatment with N-acetyl-L-cysteine, a ROS scavenger, and PD98059, an ERK inhibitor, reversed HUA-inhibited viability of cardiomyocytes. Further examination of signal transduction pathways revealed HUA-induced ROS involved in activating ERK/P38 and inhibiting PI3K/Akt in cardiomyocytes. Furthermore, the acute hyperuricaemic mouse model showed an increased phospho-ERK/p38 level in myocardial tissues. CONCLUSION: HUA induced oxidative damage and inhibited the viability of cardiomyocytes by activating ERK/p38 signalling, for a novel potential mechanism of hyperuricaemic-related cardiovascular disease.

Metformin ameliorates high uric acid-induced insulin resistance in skeletal muscle cells.

Hyperuricemia occurs together with abnormal glucose metabolism and insulin resistance. Skeletal muscle is an important organ of glucose uptake, disposal, and storage. Metformin activates adenosine monophosphate-activated protein kinase (AMPK) to regulate insulin signaling and promote the translocation of glucose transporter type 4 (GLUT4), thereby stimulating glucose uptake to maintain energy balance. Our previous study showed that high uric acid (HUA) induced insulin resistance in skeletal muscle tissue. However, the mechanism of metformin ameliorating UA-induced insulin resistance in muscle cells is unknown and we aimed to determine it. In this study, differentiated C2C12 cells were exposed to UA (15 mg/dl), then reactive oxygen species (ROS) was detected with DCFH-DA and glucose uptake with 2-NBDG. The levels of phospho-insulin receptor substrate 1 (IRS1; Ser307), phospho-AKT (Ser473) and membrane GLUT4 were examined by western blot analysis. The impact of metformin on UA-induced insulin resistance was monitored by adding Compound C, an AMPK inhibitor, and LY294002, a PI3K/AKT inhibitor. Our data indicate that UA can increase ROS production, inhibit IRS1-AKT signaling and insulin-stimulated glucose uptake, and induce insulin resistance in C2C12 cells. Metformin can reverse this process by increasing intracellular glucose uptake and ameliorating UA-induced insulin resistance.

High Uric Acid Activates the ROS-AMPK Pathway, Impairs CD68 Expression and Inhibits OxLDL-Induced Foam-Cell Formation in a Human Monocytic Cell Line, THP-1.

BACKGROUND/AIMS: Hyperuricemia is part of the metabolic-syndrome cluster of abdominal obesity, impaired glucose tolerance, insulin resistance, dyslipidemia, and hypertension. Monocytes/macrophages are critical in the development of metabolic syndrome, including gout, obesity and atherosclerosis. However, how high uric acid (HUA) exposure affects monocyte/macrophage function remains unclear. In this study, we investigated the molecular mechanism of HUA exposure in monocytes/macrophages and its impact on oxidized low-density lipoprotein (oxLDL)-induced foam-cell formation in a human monocytic cell line, THP-1. METHODS: We primed THP-1 cells with phorbol-12-myristate-13-acetate (PMA) for differentiation, then exposed cells to HUA and detected the production of reactive oxygen species (ROS) and analyzed the level of phospho-AMPKα. THP-1 cells were pre-incubated with Compound C, an AMPK inhibitor, or N-acetyl-L-cysteine (NAC), a ROS scavenger, or HUA before PMA, to assess CD68 expression and phospho-AMPKα level. PMA-primed THP-1 cells were pre-treated with oxLDL before Compound C and HUA treatment. Western blot analysis was used to examine the levels of phospho-AMPKα, CD68, ABCG1, ABCA1, cyclooxygenase-2 (COX-2) and NF-κB (p65). Flow cytometry was used to assess ROS production and CD68 expression in live cells. Oil-red O staining was used to observe oxLDL uptake in cells. RESULTS: HUA treatment increased ROS production in PMA-primed THP-1 cells; NAC blocked HUA-induced oxidative stress. HUA treatment time-dependently increased phospho-AMPKα level in PMA-primed THP-1 cells. The HUA-induced oxidative stress increased phospho-AMPKα levels, which was blocked by NAC. HUA treatment impaired CD68 expression during cell differentiation by activating the AMPK pathway, which was reversed by Compound C treatment. Finally, HUA treatment inhibited oxLDL uptake in the formation of foam cells in THP-1 cells, which was blocked by Compound C treatment. HUA treatment significantly increased the expression of ABCG1 and reversed the oxLDL-reduced ABCG1 expression but did not affect the expression of ABCA1, NF-κB (p65) or COX-2. CONCLUSIONS: HUA exposure activated the ROS-AMPK pathway, impaired CD68 expression, and inhibited oxLDL-induced foam-cell formation in a human monocytic cell line, THP-1.

Metformin inhibits estrogen-dependent endometrial cancer cell growth by activating the AMPK-FOXO1 signal pathway.

Metformin is an oral biguanide commonly used for treating type II diabetes and has recently been reported to possess antiproliferative properties that can be exploited for the prevention and treatment of a variety of cancers. The mechanisms underlying this effect have not been fully elucidated. Our study shows a marked loss of AMP-activated protein kinase (AMPK) phosphorylation and nuclear human Forkhead box O1 (FOXO1) protein in estrogen-dependent endometrial cancer (EC) tumors compared to normal control endometrium. Metformin treatment suppressed EC cell growth in a time-dependent manner in vitro; this effect was cancelled by cotreatment with an AMPK inhibitor, compound C. Metformin decreased FOXO1 phosphorylation and increased FOXO1 nuclear localization in Ishikawa and HEC-1B cells, with non-significant increase in FOXO1 mRNA expression. Moreover, compound C blocked the metformin-induced changes of FOXO1 and its phosphorylation protein, suggesting that metformin upregulated FOXO1 activity by AMPK activation. Similar results were obtained after treatment with insulin. In addition, transfection with siRNA for FOXO1 cancelled metformin-inhibited cell growth, indicating that FOXO1 mediated metformin to inhibit EC cell proliferation. A xenograft mouse model further revealed that metformin suppressed HEC-1B tumor growth, accompanied by downregulated ki-67 and upregulated AMPK phosphorylation and nuclear FOXO1 protein. Taken together, these data provide a novel mechanism of antineoplastic effect for metformin through the regulation of FOXO1, and suggest that the AMPK-FOXO1 pathway may be a therapeutic target to the development of new antineoplastic drugs.

AMPD1 regulates mTORC1-p70 S6 kinase axis in the control of insulin sensitivity in skeletal muscle.

BACKGROUND: Insulin resistance triggered by excess fat is a key pathogenic factor that promotes type 2 diabetes. Understanding molecular mechanisms of insulin resistance may lead to the identification of a novel therapeutic target for type 2 diabetes. AMPD1, an isoform of AMP deaminase (AMPD), is suggested to play roles in the regulation of glucose metabolism through controlling AMP-activated protein kinase (AMPK) activation. We reported that the diet-induced insulin resistance was improved in AMPD1-deficient mice compared to wild type mice. To further delineate this observation, we studied changes of insulin signaling in skeletal muscle of wild type (WT) and AMPD1-deficient mice. METHODS: Phosphorylation levels of kinases and expression levels of mTOR components were quantified by immunoblotting using protein extracts from tissues. The interaction between mTOR and Raptor was determined by immunoblotting of mTOR immunoprecipitates with anti-Raptor antibody. Gene expression was studied by quantitative PCR using RNA extracted from tissues. RESULTS: Phosphorylation levels of AMPK, Akt and p70 S6 kinase in skeletal muscle were higher in AMPD1-deficient mice compared to WT mice after high fat diet challenge, while they did not show such difference in normal chow diet. Also, no significant changes in phosphorylation levels of AMPK, Akt or p70 S6 kinase were observed in liver and white adipose tissue between WT and AMPD1-deficient mice. The expression levels of mTOR, Raptor and Rictor tended to be increased by AMPD1 deficiency compared to WT after high fat diet challenge. AMPD1 deficiency increased Raptor-bound mTOR in skeletal muscle compared to WT after high fat diet challenge. Gene expression of peroxisome proliferator-activated receptor-γ coactivator 1α and ß, downstream targets of p70 S6 kinase, in skeletal muscles was not changed significantly by AMPD1 deficiency compared to the wild type after high fat diet challenge. CONCLUSION: These data suggest that AMPD1 deficiency activates AMPK/Akt/mTORC1/p70 S6 kinase axis in skeletal muscle after high fat diet challenge, but not in normal chow diet. These changes may contribute to improve insulin resistance.

High uric acid directly inhibits insulin signalling and induces insulin resistance.

BACKGROUND AND AIM: Accumulating clinical evidence suggests that hyperuricemia is strongly associated with abnormal glucose metabolism and insulin resistance. However, how high uric acid (HUA) level causes insulin resistance remains unclear. We aimed to determine the direct role of HUA in insulin resistance in vitro and in vivo in mice. METHODS: An acute hyperuricemia mouse model was created by potassium oxonate treatment, and the impact of HUA level on insulin resistance was investigated by glucose tolerance test, insulin tolerance test and insulin signalling, including phosphorylation of insulin receptor substrate 1 (IRS1) and Akt. HepG2 cells were exposed to HUA treatment and N-acetylcysteine (NAC), reactive oxygen species scavenger; IRS1 and Akt phosphorylation was detected by Western blot analysis after insulin treatment. RESULTS: Hyperuricemic mice showed impaired glucose tolerance with insulin resistance. Hyperuricemia inhibited phospho-Akt (Ser473) response to insulin and increased phosphor-IRS1 (Ser307) in liver, muscle and fat tissues. HUA induced oxidative stress, and the antioxidant NAC blocked HUA-induced IRS1 activation and Akt inhibition in HepG2 cells. CONCLUSION: This study supplies the first evidence of HUA directly inducing insulin resistance in vivo and in vitro. Increased uric acid level may inhibit IRS1 and Akt insulin signalling and induce insulin resistance. The reactive oxygen species pathway plays a key role in HUA-induced insulin resistance.

AMP-activated protein kinase suppresses the in vitro and in vivo proliferation of hepatocellular carcinoma.

AMP-activated protein kinase (AMPK) is a central metabolic sensor and plays an important role in regulating glucose, lipid and cholesterol metabolism. Therefore, AMPK is a key therapeutic target in diabetes. Recent pilot studies have suggested that diabetes drugs may reduce the risk of cancer by affecting the AMPK pathway. However, the association between AMPK and the proliferation of hepatocellular carcinoma (HCC) is unknown. In this study, we investigated the relationship between AMPK activity and the proliferation of HCC in cell lines, nude mice and human clinic samples. We first investigated the relationship between AMPK activity and cell proliferation in two HCC cell lines, PLC/PRF/5 and HepG2, by two AMPK activators, 5-aminoimidazole-4-carboxamide-1-h-D-ribofuranoside (AICAR) and metformain. AICAR and metformin treatment significantly inhibited the proliferation of HCC cells and induced cell cycle arrest at G1-S checkpoint. We then observed that metformin abrogated the growth of HCC xenografts in nude mice. The clinical pathology of AMPK activity in HCC, including cell proliferation, differential grade, tumor size and microvessel density, was studied by using 30 clinical tissue samples. In HCC tissue samples, phosphorylated AMPK was expressed mainly in cytoplasm. AMPK activity decreased significantly in HCC in comparison with paracancerous liver tissues (P<0.05). AMPK activity was negatively correlated with the level of Ki-67 (a marker of cell proliferation), differential degradation and tumor size (P<0.05), but not with microvessel density, hemorrhage or necrosis in HCC. Our findings suggest that AMPK activity inhibits the proliferation of HCC and AMPK might be an effective target for prevention and treatment of HCC.