Clinical studies have shown hyperuricemia strongly associated with insulin resistance as well as cardiovascular disease. Direct evidence of how high uric acid (HUA) affects insulin resistance in ...cardiomyocytes, but the pathological mechanism of HUA associated with cardiovascular disease remains to be clarified. We aimed to examine the effect of HUA on insulin sensitivity in cardiomyocytes and on insulin resistance in hyperuricemic mouse model. We exposed primary cardiomyocytes and a rat cardiomyocyte cell line, H9c2 cardiomyocytes, to HUA, then quantified glucose uptake with a fluorescent glucose analog, 2-NBDG, after insulin challenge and detected reactive oxygen species (ROS) production. Western blot analysis was used to examine the levels of insulin receptor (IR), phosphorylated insulin receptor substrate 1 (IRS1, Ser307) and phospho-Akt (Ser473). We monitored the impact of HUA on insulin resistance, insulin signaling and IR, phospho-IRS1 (Ser307) and phospho-Akt levels in myocardial tissue of an acute hyperuricemia mouse model established by potassium oxonate treatment. HUA inhibited insulin-induced glucose uptake in H9c2 and primary cardiomyocytes. It increased ROS production; pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger, reversed HUA-inhibited glucose uptake induced by insulin. HUA exposure directly increased the phospho-IRS1 (Ser307) response to insulin and inhibited that of phospho-Akt in H9C2 cardiomyocytes, which was blocked by NAC. Furthermore, the acute hyperuricemic mice model showed impaired glucose tolerance and insulin tolerance accompanied by increased phospho-IRS1 (Ser307) and inhibited phospho-Akt response to insulin in myocardial tissues. HUA inhibited insulin signaling and induced insulin resistance in cardiomyocytes in vitro and in vivo, which is a novel potential mechanism of hyperuricemic-related cardiovascular disease.
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.
Atherosclerotic vascular disease (ASVD) is the leading cause of death worldwide. Hyperuricemia is the fourth risk factor for atherosclerosis after hypertension, diabetes, and hyperlipidemia. The ...mechanism of hyperuricemia affecting the occurrence and development of atherosclerosis has not been fully elucidated. Mononuclear macrophages play critical roles in all stages of atherosclerosis. Studies have confirmed that both hyperuricemia and ferroptosis promote atherosclerosis, but whether high level of uric acid (HUA) promotes atherosclerosis by regulating ferroptosis in macrophages remains unclear. We found that HUA significantly promoted the development of atherosclerotic plaque and downregulated the protein level of the NRF2/SLC7A11/GPX4 signaling pathway in ApoE−/− mice. Next, we evaluated the effect of HUA and ferroptosis inhibitor ferrostatin-1 (Fer-1) treatment on the formation of macrophage-derived foam cells. HUA promoted the formation of foam cells, decreased cell viability, and increased iron accumulation and lipid peroxidation in macrophages treated with oxidized low-density lipoprotein (oxLDL); these effects were reversed by Fer-1 treatment. Mechanistically, HUA significantly inhibited autophagy and the protein level of the NRF2/SLC7A11/GPX4 signaling pathway. Fer-1 activated autophagy and upregulated the level of ferroptosis-associated proteins. Moreover, an NRF2 inducer (tertbutyl hydroquinone (TBHQ)) and autophagy activator (rapamycin (RAPA)) could reverse the inhibitory effect of HUA on foam cell survival. Our results suggest that HUA-induced ferroptosis of macrophages is involved in the formation of atherosclerotic plaques. More importantly, enhancing autophagy and inhibiting ferroptosis by activating NRF2 may alleviate HUA-induced atherosclerosis. These findings might contribute to a deeper understanding of the role of HUA in the pathogenesis of atherosclerosis and provide a therapeutic target for ASVD associated with hyperuricemia.
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.
Metformin may exert its anti‐proliferative effect on EC cells by activating AMPK and thus decreasing phosphorylation of FOXO1 protein, thereby triggering the relocalization of FOXO1 protein from the cytoplasm to the nucleus and resulting in increased FOXO1 activity. This study provide a novel mechanism of anti‐neoplastic effect for metformin through the regulation of FOXO1, and suggest that AMPK‐FOXO1 pathway may be a therapeutic target to the development of new anti‐neoplastic drugs.
Background:Hyperuricemia induces endothelial dysfunction, oxidative stress and inflammation, increasing cardiovascular morbidities. It also raises the incidence of atrial fibrillation; however, ...underlying mechanisms are unknown.Methods and Results:The effects of urate on expression of Kv1.5 in cultured mouse atrial myocytes (HL-1 cells) using reverse transcriptase-PCR, immunoblots, flow cytometry and patch-clamp experiments were studied. Treatment with urate at 7 mg/dl for 24 h increased the Kv1.5 protein level, enhanced ultra-rapid delayed-rectifier K+channel currents and shortened action potential duration in HL-1 cells. HL-1 cells expressed the influx uric acid transporter (UAT), URATv1, and the efflux UATs, ABCG2 and MRP4. An inhibitor against URATv1, benzbromarone, abolished the urate effects, whereas an inhibitor against ABCG2, KO143, augmented them. Flow cytometry showed that urate induced an increase in reactive oxygen species, which was abolished by the antioxidant, N-acetylcysteine (NAC), and the NADPH-oxidase inhibitor, apocynin. Both NAC and apocynin abolished the enhancing effects of urate on Kv1.5 expression. A urate-induced increase in the Kv1.5 proteins was accompanied by phosphorylation of extracellular signal-regulated kinase (ERK), and was abolished by an ERK inhibitor, PD98059. NAC abolished phosphorylation of ERK by urate.Conclusions:Intracellular urate taken up by UATs enhanced Kv1.5 protein expression and function in HL-1 atrial myocytes, which could be attributable to ERK phosphorylation and oxidative stress derived from nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase. (Circ J 2015; 79: 2659–2668)
Aims: Acute rupture or erosion of unstable atherosclerotic plaques is a major cause of adverse consequences of atherosclerotic cardiovascular disease, often leading to myocardial infarction or ...stroke. High uric acid (HUA) is associated with the increasing risk of cardiovascular events and death. However, the mechanism by which HUA promotes atherosclerosis and whether HUA affects plaque stability are still unclear.Methods: We constructed an atherosclerotic Apoe−/− mouse model with HUA. The progression of atherosclerosis and plaques was determined by Oil Red O staining, hematoxylin and eosin (H&E) staining, and Masson staining. TdT-mediated dUTP nick-end labeling assay and immunohistochemistry were used to observe the changes of apoptosis and autophagy in plaques, respectively. Then, we validated the in vivo results with RAW 264.7 cell line.Results: HUA promoted atherosclerosis and exacerbated plaque vulnerability, including significantly increased macrophage infiltration, lipid accumulation, enlarged necrotic cores, and decreased collagen fibers. HUA increased cell apoptosis and inhibited autophagy in plaques. In vitro results showed that HUA decreased cell viability and increased cell apoptosis in foam cells macrophages treated with oxidized low-density lipoprotein. An activator of autophagy, rapamycin, can partially reverse the increasing apoptosis.Conclusion: HUA promoted atherosclerosis and exacerbated plaque vulnerability, and HUA facilitates foam cell apoptosis by inhibiting autophagy.
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.
Acute ischaemic stroke (AIS) is a major cause of disability and death, which is a serious threat to human health and life. Wasp venom extracted from Vespa magnifica Smith (Vespidae) could treat major ...neurological disorders.
This study investigated the effects of wasp venom on AIS in rats.
We used a transient middle cerebral artery occlusion (MCAO) model in Sprague-Dawley rats (260-280 g, n = 8-15) with a sham operation group being treated as negative control. MCAO rats were treated with wasp venom (0.05, 0.2 and 0.6 mg/kg, i.p.) using intraperitoneal injection. After treatment 48 h, behavioural tests, cortical blood flow (CBF), TTC staining, H&E staining, Nissl staining, TUNEL assay, immunohistochemistry (IHC) and ELISA were employed to investigate neuroprotective effects of wasp venom.
Compared with the MCAO group, wasp venom (0.6 mg/kg) improved neurological impairment, accelerated CBF recovery (205.6 ± 52.92 versus 216.7 ± 34.56), reduced infarct volume (337.1 ± 113.2 versus 140.7 ± 98.03) as well as BBB permeability as evidenced by changes in claudin-5 and AQP4. In addition, function recovery of stroke by wasp venom treatment was associated with a decrease in TNF-α, IL-1β, IL-6 and inhibition activated microglia as well as apoptosis. Simultaneously, the wasp venom regulated the angiogenesis factors VEGF and b-FGF in the brain.
Wasp venom exhibited a potential neuroprotective effect for AIS. In the future, we will focus on determining whether the observed actions were due to a single compound or the interaction of multiple components of the venom.
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.
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.
•Molecular mechanism.•Metformin effect.•A new possible therapy for Hyperuricemia.
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