Phosphorylation Levels Of AMP-activated Protein Kinase Research Articles

Extracellular Matrix Stiffness Induces Mitochondrial Morphological Heterogeneity via AMPK Activation

To investigate the mechanical responses of mitochondrial morphology to extracellular matrix stiffness in human mesenchymal stem cells (hMSCs) and the role of AMP-activated protein kinase (AMPK) in the regulation of mitochondrial mechanoresponses. Two polyacrylamide (PAAm) hydrogels, a soft one with a Young's modulus of 1 kPa and a stiff one of 20 kPa, were prepared by changing the monomer concentrations of acrylamide and bis-acrylamide. Then, hMSCs were cultured on the soft and stiff PAAm hydrogels and changes in mitochondrial morphology were observed using a laser confocal microscope. Western blot was performed to determine the expression and activation of AMPK, a protein associated with mitochondrial homeostasis. Furthermore, the activation of AMPK was regulated on the soft and stiff matrixes by AMPK activator A-769662 and the inhibitor Compound C, respectively, to observe the morphological changes of mitochondria. The morphology of the mitochondria in hMSCs showed heterogeneity when there was a change in gel stiffness. On the 1 kPa soft matrix, 74% mitochondria exhibited a dense, elongated filamentous network structure, while on the 20 kPa stiff matrix, up to 63.3% mitochondria were fragmented or punctate and were sparsely distributed. Western blot results revealed that the phosphorylated AMPK (p-AMPK)/AMPK ratio on the stiff matrix was 1.6 times as high as that on the soft one. Immunofluorescence assay results revealed that the expression of p-AMPK was elevated on the hard matrix and showed nuclear localization, which indicated that the activation of intracellular AMPK increased continuously along with the increase in extracellular matrix stiffness. When the hMSCs on the soft matrix were treated with A-769662, an AMPK activator, the mitochondria transitioned from a filamentous network morphology to a fragmented morphology, with the ratio of filamentous network decreasing from 74% to 9.5%. Additionally, AMPK inhibition with Compound C promoted mitochondrial fusion on the stiff matrix and significantly reduced the generation of punctate mitochondria. Extracellular matrix stiffness regulates mitochondrial morphology in hMSCs through the activation of AMPK. Stiff matrix promotes the AMPK activation, resulting in mitochondrial fission and the subsequent fragmentation of mitochondria. The impact of matrix stiffness on mitochondrial morphology can be reversed by altering the level of AMPK phosphorylation.

Improved endurance capacity of diabetic mice during SGLT2 inhibition: Role of AICARP, an AMPK activator in the soleus.

Diabetes is associated with an increased risk of deleterious changes in muscle mass and function or sarcopenia, leading to physical inactivity and worsening glycaemic control. Given the negative energy balance during sodium-glucose cotransporter-2 (SGLT2) inhibition, whether SGLT2 inhibitors affect skeletal muscle mass and function is a matter of concern. However, how SGLT2 inhibition affects the skeletal muscle function in patients with diabetes remains insufficiently explored. We aimed to explore the effects of canagliflozin (CANA), an SGLT2 inhibitor, on skeletal muscles in genetically diabetic db/db mice focusing on the differential responses of oxidative and glycolytic muscles. Db/db mice were treated with CANA for 4weeks. We measured running distance and handgrip strength to assess skeletal muscle function during CANA treatment. At the end of the experiment, we performed a targeted metabolome analysis of the skeletal muscles. CANA treatment improved the reduced endurance capacity, as revealed by running distance in db/db mice (414.9±52.8 vs. 88.7±22.7m, P<0.05). Targeted metabolome analysis revealed that 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl 5'-monophosphate (AICARP), a naturally occurring AMP-activated protein kinase (AMPK) activator, increased in the oxidative soleus muscle (P<0.05), but not in the glycolytic extensor digitorum longus muscle (P=0.4376), with increased levels of AMPK phosphorylation (P<0.01). This study highlights the potential role of the AICARP/AMPK pathway in oxidative rather than glycolytic skeletal muscles during SGLT2 inhibition, providing novel insights into the mechanism by which SGLT2 inhibitors improve endurance capacity in patients with type 2 diabetes.

The Maintenance of AMPK Activity Eliminates Abnormally Accelerated Differentiation of Primary Myoblasts Isolated from Atrophied Rat Soleus Muscle

Mechanical unloading of skeletal muscles leads to the development of atrophic processes and a decrease in the total number of satellite cells (SCs) that are involved in muscle regeneration. In vitro studies revealed an increased differentiation of myoblasts derived from rat soleus muscle after an unloading-induced decrease in AMP-activated protein kinase (AMPK). AMPK is necessary for the activation of SCs and also participates in the regulation of myoblast proliferation and differentiation. It can be assumed that a decrease in the activity of AMPK after mechanical unloading can contribute to the acceleration of myoblast differentiation. The main purpose of this study was to elucidate a possible role of AMPK in the regulation of differentiation of myoblasts isolated from rat soleus muscle after mechanical unloading. To test this hypothesis, a specific AMPK activator, AICAR, was used to prevent a decrease in AMPK activity during differentiation of myoblasts isolated from rat soleus muscle after 7-day unloading. Immunocytochemistry, PCR-RT and Western blotting were used to assess changes during myoblast differentiation. In differentiating myoblasts derived from the unloaded soleus muscle there was a significant decrease in AMPK (Thr172) and ACC (Ser 79) phosphorylation levels, an increase in myotube differentiation index, myoblast fusion factors and the expression of myogenic regulatory factors (MRF). Furthermore, there was a decrease in the expression of slow myosin heavy chains (MyHC) and an increase in the expression of fast MyHC isoforms. AICAR treatment of differentiating myoblasts obtained from the unloaded soleus muscle prevented a decrease in AMPK and ACC phosphorylation, returned the expression levels of MRF and fast isoforms of MyHC to the control levels as well as maintained the expression of slow MyHC. Thus, abnormally accelerated differentiation of myoblasts isolated from atrophied rat soleus muscle can be compensated by maintaining the control levels of AMPK activity using AICAR.

AMP-activated protein kinase-farnesoid X receptorpathway contributes to oleanolic acid-induced liver injury.

Natural pentacyclic triterpenoid oleanolic acid (OA) is used as an over-the-counter drug for acute and chronic hepatitis. However, clinical use of OA-containing herbal medicines has been reported to cause cholestasis, and the specific mechanism is unknown. The purpose of this study was to explore how OA causes cholestatic liver injury via the AMP-activated protein kinase (AMPK)-farnesoid X receptor (FXR) pathway. In animal experiments, it was found that OA treatment activated AMPK and decreased FXR and bile acid efflux transport proteins expression. When intervened with the specific inhibitor Compound C (CC), it was observed that AMPK activation was inhibited, the reduction of FXR and bile acid efflux transport protein expression was effectively alleviated, serum biochemical indicators were significantly reduced, and liver pathological damage brought about by OA was effectively ameliorated. In addition, OA was found to downregulate the expression of FXR and bile acid efflux transport proteins by activating the ERK1/2-LKB1-AMPK pathway in cellular experiments. The ERK1/2 inhibitor U0126 was used to pretreat primary hepatocytes, and this drastically reduced the phosphorylation levels of LKB1 and AMPK. The inhibition effects of OA on FXR and bile acid efflux transport proteins were also effectively alleviated after pretreatment with CC. In addition, OA-induced downregulation of FXR gene and protein expression levels was significantly prevented after silencing AMPKα1 expression in AML12 cells. Our study demonstrated that OA inhibited FXR and bile acid efflux transporters through the activation of AMPK, thus leading to cholestatic liver injury.

Exercise-induced FKBP8-mediated Mitophagy Via AMPK Activation In Soleus Muscle Of Rats

Scientific Abstract PURPOSE: To investigate the effects of downhill running on mitochondrial structure/function and expression levels of mitophagy-related proteins in rat skeletal muscle, and whether AMP-activated protein kinase (AMPK) plays a regulatory role in this context. METHODS: Male Sprague-Dawley rats were divided into a control (C, n = 8) or an exercise (E, n = 56) group. Rats in the E group were exercised on a treadmill down a 16° incline at 16 m/min for 90 min, and were further divided into 0 h (E0), 12 h (E12), 24 h (E24), 48 h (E48) and 72 h (E72) post-exercise sub-groups. Rats in the E0 and E12 sub-groups received acute AMPK agonist AICAR treatment (500 mg/kg via I.P.) or saline (n = 8 each) 30 min before exercise. At each time point the soleus muscle was collected under full anesthesia. Mitochondrial ultrastructural changes in skeletal muscle were observed by using a transmission electron microscope. Protein expression levels of skeletal muscle p-AMPKα1/2(T172), total AMPKα, heat shock protein 60 (HSP60), cyclophilin D (CypD), mitochondrial FK506 binding protein 8 (FKBP8/FKBP38), microtubule-associated protein 1 light chain 3 (LC3) and cytosolic cytochrome C (Cyt C) were determined by using Western blot. LC3 co-localizations with mitochondria and FKBP8 were measured by the immunofluorescence technique. One-way and two-way ANOVAs were used for data analysis. RESULTS: After downhill running, skeletal muscle mitochondrial structure appeared to be abnormal and contained a large number of mitophagosomes; the expression levels of AMPK phosphorylation (E12 = 1.81 ± 0.54), FKBP8 (E12 = 3.11 ± 0.26), LC3 (E12 = 1.80 ± 0.12), CypD (E0 = 1.41 ± 0.26) and Cyt C (E0 = 2.31 ± 0.68) and the co-localization of FKBP8 and LC3 were significantly higher, whereas the expression levels of mitochondrial quantitative protein HSP60 (E12 = 0.62 ± 0.09) were significantly lower than those in the C group (p < 0.05 to p < 0.01). AICAR treatment enhanced AMPK phosphorylation in exercised muscle and the expression levels of mitochondrial FKBP8 and LC3 involved in mitophagy. CONCLUSION: A session of downhill running activated the AMPK phosphorylation and LC3 co-localizations with mitochondria and FKBP8, and induced mitophagy and mitochondrial damage within rat skeletal muscle. (Supported by Natural Science Foundation of China grant 31900842).

AMP-activated protein kinase mediates glycolysis in post-mortem breast muscle of broilers

The aim of this study was to confirm the role of AMP-activated protein kinase (AMPK) in the regulation of post-mortem breast muscle glycolysis in the broiler, we evaluated the effects of intraperitoneal injection of the specific AMPK activator (5-aminoimidazole-4-carboxamide ribonucleoside, AICAR) and inhibitor (6-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a] pyrimidine, Compound C) on AMPK phosphorylation (p-AMPK) level, meat quality, glycolytic metabolites and glycolytic enzymes activities in post-mortem breast muscle of broilers. Compared to the CON group, intraperitoneal injection of AIRCR increased p-AMPK level, glycolytic enzyme activities and lactate content in post-mortem breast muscle, increased lightness (L*) and drip loss, but decreased pH45min and pH24h. However, intraperitoneal injection of Compound C, decreased p-AMPK level, glycolytic enzyme activities, glucose-6-phosphate and lactate content in post-mortem breast muscle, and increased pH24h. Based on these results, we conclude that AMPK played a vital role in regulating post-mortem glycolysis in the breast muscle of broilers. HIGHLIGHTS Injection of AIRCR promoting muscle glycolysis. Injection of Compound C alleviating muscle glycolysis. AMPK played a vital role in regulating post-mortem muscle glycolysis.

Salvianolic acid A promotes mitochondrial biogenesis and function via regulating the AMPK/PGC-1α signaling pathway in HUVECs.

Mitochondrial dysregulation is an important pathology that leads to endothelial dysfunction, and the occurrence and development of cardiovascular diseases. Salvianolic acid A (SAA) has been demonstrated to be effective in the treatment of vascular complications of type 2 diabetes mellitus. Limited information has been reported on the effects of SAA on mitochondrial function in endothelial cells. In the present study, the effects of SAA on mitochondrial biogenesis and the related underlying mechanisms were investigated in human umbilical vein endothelial cells (HUVECs). Mitotracker red staining and transmission electron microscopy were used to evaluate the effect of SAA on mitochondrial quality. The effect of SAA treatment on mitochondrial DNA/nuclear DNA ratio of HUVECs was detected by real-time quantitative PCR. Western blot was used to determine the protein expression levels of complex III and Complex IV of mitochondrial oxidative phosphorylation subunit, and ATP production was determined by ATP test kit. Real-time quantitative PCR and Western blot were used to determine the effects of SAA on the expression of peroxisome proliferator-activated receptor γ coactivator (PGC-1α) and its target genes nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM) proteins and genes. Finally, in the presence of 5'AMP-activated protein kinase (AMPK) specific inhibitors, the expression of PGC-1α, NRF1 and TFAM proteins and the phosphorylation levels of AMPK and Acetyl CoA Carboxylase (ACC) were detected by Western blot or real-time quantitative PCR. The results showed that SAA treatment significantly promoted mitochondrial biogenesis and enhanced mitochondrial function of HUVECs. SAA significantly increased the expression levels of PGC-1α and its target genes NRF1 and (TFAM), a key regulator of mitochondrial biogenesis in HUVECs. These enhancements were accompanied by significantly increased phosphorylation of AMPK and ACC, and were significantly inhibited by specific AMPK inhibitors. These results suggest that SAA may promote mitochondrial biogenesis in endothelial cells by activating the AMPK-mediated PGC-1α/TFAM signaling pathway. These data provide new insights into the mechanism of action of SAA in treating diabetic vascular complications.

Anti‐obesity effect of Premature Citrus Extract in high‐fat diet induced obesity mice

BackgroundObesity is a worldwide health threat. Premature citrus extract (PCE) are known to contain high concentrations of flavonoids, polyphenols, and organic acids with health functions. The current study aimed to investigate the anti‐obesity effect of PCE on a high‐fat diet (HFD)‐induced obesity mice.MethodMice were fed on chow or HFD with or without Orlistat (ORL), Hesperidin (HES), and PCE treatment for 10 weeks. Triglyceride (TG) content and total cholesterol (TC), and low‐density lipoprotein cholesterol (LDL‐c), and high‐density lipoprotein cholesterol (HDL‐c) levels were determined by TG, HDL and LDL Assay Kit (BioAssay System). The phosphorylation levels of AMP‐activated protein kinase (AMPK) and acetyl Co‐A carboxylase (ACC); the expression of sterol regulatory element binding protein‐2 (SREBP‐2), 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase (HMGCR), Acyl‐coenzyme A: cholesterol acyltransferases (ACAT‐1) and mitochondrial uncoupling proteins (UCP‐3) were observed via western blot analysis.ResultsPCE and HES administered group significantly reduced the weights of body, liver, and white adipose tissue compared to the HFD group. Supplementation with PCE and HES significantly decreased the levels of TG, TC, and (LDL‐c), and increased (HDL‐c) levels in serum compared to the HFD group. Furthermore, treatment with PCE and HES significantly decreased the levels of TG and TC in the liver whereas increased the levels of TG and TC in feces compared to the HFD group. Western blot analysis revealed that PCE and HES increased the phosphorylation levels of both AMPK and ACC and significantly decreased the expression levels of SREBP‐2, HMGCR, and ACAT‐1 in the liver compared to the HFD group. In addition, both PCE and HES increased the expression level of UCP‐3 in skeletal muscle tissue.ConclusionOverall results suggest that PCE and HES exerts an anti‐obesity effect via activation of AMPK and associated signaling cascades related to lipid metabolism in HFD‐induced obesity mice

Anti-adipogenic effect of the flavonoids through the activation of AMPK in palmitate (PA)-treated HepG2 cells.

BackgroundFlavonoids are natural polyphenols found widely in citrus fruit and peel that possess anti-adipogenic effects. On the other hand, the detailed mechanisms for the anti-adipogenic effects of flavonoids are unclear.ObjectivesThe present study observed the anti-adipogenic effects of five major citrus flavonoids, including hesperidin (HES), narirutin (NAR), nobiletin (NOB), sinensetin (SIN), and tangeretin (TAN), on AMP-activated protein kinase (AMPK) activation in palmitate (PA)-treated HepG2 cells.MethodsThe intracellular lipid accumulation and triglyceride (TG) contents were quantified by Oil-red O staining and TG assay, respectively. The glucose uptake was assessed using 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) assay. The levels of AMPK, acetyl-CoA carboxylase (ACC), and glycogen synthase kinase 3 beta (GSK3β) phosphorylation, and levels of sterol regulatory element-binding protein 2 (SREBP-2) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) expression were analyzed by Western blot analysis. The potential interaction between the flavonoids and the γ-subunit of AMPK was investigated by molecular docking analysis.ResultsThe flavonoid treatment reduced both intracellular lipid accumulation and TG content in PA-treated HepG2 cells significantly. In addition, the flavonoids showed increased 2-NBDG uptake in an insulin-independent manner in PA-treated HepG2 cells. The flavonoids increased the AMPK, ACC, and GSK3β phosphorylation levels and decreased the SREBP-2 and HMGCR expression levels in PA-treated HepG2 cells. Molecular docking analysis showed that the flavonoids bind to the CBS domains in the regulatory γ-subunit of AMPK with high binding affinities and could serve as potential AMPK activators.ConclusionThe overall results suggest that the anti-adipogenic effect of flavonoids on PA-treated HepG2 cells results from the activation of AMPK by flavonoids.

APPL1 ameliorates myocardial ischemia-reperfusion injury by regulating the AMPK signaling pathway.

Myocardial ischemia-reperfusion injury results in elevated reactive oxygen species (ROS) production and causes oxidative stress damage. Therefore, the current study aimed to investigate whether adaptor protein phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) could induce the expression of antioxidant enzymes through AMP-activated protein kinase (AMPK) signaling in order to alleviate the injury caused by ischemia/hypoxia-reperfusion. Following induction of hypoxia-reoxygenation (H/R) injury in H9c2 cells, the liver kinase B1 (LKB1)/AMPK/acetyl-CoA carboxylase α (ACC) signaling pathway was investigated using western blot analysis, along with the detection of superoxide dismutase (SOD)2 and SOD3 expression. Additionally, cell viability was detected using a Cell Counting Kit-8 assay and ROS production was analyzed using ROS staining, whereas the expression levels of inflammatory mediators (TNF-α, monocyte chemoattractant protein 1 and IL-1β), apoptosis mediators [cleaved caspase-3, cleaved poly (ADP-ribose) polymerase and Bcl-2] and nuclear factor erythroid 2-related factor 2 signaling pathway-related proteins were detected via western blot analysis following overexpression of APPL1 alone or in combination with compound C treatment (an AMPK inhibitor). The results indicated that H/R induction upregulated the phosphorylation levels of LKB1, AMPK and ACC, and decreased the expression levels of APPL1 and SOD enzyme activities. APPL1 overexpression increased the phosphorylation levels of LKB1, AMPK and ACC, SOD enzyme activity and cell viability whereas the expression levels of proinflammatory mediators and proapoptotic mediators, and the levels of ROS production were markedly decreased when compared with H/R group with empty plasmid transfection. APPL overexpression-mediated effects were significantly abrogated by compound C. Taken together, the data indicated that APPL1 inhibited ROS production and H/R-induced myocardial injury via the AMPK signaling pathway. Therefore, APPL1 may serve as a potential therapeutic target for myocardial H/R injury.

AMP-activated protein kinase contributes to myofibrillar protein hydrolysis in bovine skeletal muscle through postmortem mitochondrial dysfunction-induced apoptosis.

This study aimed to verify the role of AMP-activated protein kinase (AMPK) in mitochondrial dysfunction-induced apoptosis and postmortem bovine muscle tenderization. AMPK phosphorylation levels, mitochondrial dysfunction, mitochondrial apoptotic factors, and myofibrillar protein hydrolysis were assessed in the control group and Compound C (AMPK inhibitor) group over a 168hr aging period. Compared with the Compound C group, the control group had an extremely significantly increased AMPK activity at 6-120hr (p<.01) and a 62.3% and 42.1% higher mitochondrial Bax/Bcl-2 ratio at 6 and 12hr, respectively (p<.05). Moreover, the control group had a significantly or extremely significantly higher mitochondrial dysfunction and cytoplasmic cytochrome c content at 6-72 and 12-72hr, respectively (p<.05, p<.01); a 23.2%, 26.5%, and 26.1% increased caspase-3 expression levels at 12, 24, and 72hr, respectively (p<.05); a significantly higher proportion of apoptotic nuclei at 24-168hr (p<.05); and a 30.8%, 35.8%, 43.9%, and 39.5% increased production of 45-, 38-, 36-, 30-, and 28-kDa proteins at 168hr, respectively (p<.05). Taken together, these results suggested that activated AMPK promoted mitochondrial apoptosis and bovine muscle tenderization during postmortem aging by increasing the Bax/Bcl-2 ratio on the mitochondrial membrane. PRACTICAL APPLICATIONS: Based on consumer preference, chilled fresh meat is gradually becoming the future trend of the meat industry. Poorly tenderized beef often affects consumers' desire to make secondary purchases and leads to large losses to the meat industry. Therefore, AMP-activated protein kinase, which regulates postmortem mitochondrial apoptosis and bovine muscle tenderization, is a valid research target.

MicroRNA-506-3p targets SIRT1 and suppresses AMPK pathway activation to promote hepatic steatosis.

Nonalcoholic fatty liver disease (NAFLD) is a complex type of liver disease that represents an important global health threat. The mechanistic basis of this disease remains incompletely understood. The present study sought to explore whether microRNA (miR)-506-3p served a functional role in the onset and/or progression of NAFLD. To that end, high levels of glucose were used to treat liver cancer cell lines (HepG2 and Huh7) to model hepatic steatosis, and the expression levels of miR-506-3p and its downstream target genes were assessed. The cells of this hepatic steatosis model were transfected with miR-506-3p mimic molecules to explore the effect of miR-506-3p overexpression on cell viability, target gene expression and AMP-activated protein kinase (AMPK) phosphorylation. Via bioinformatics approaches, sirtuin 1 (SIRT1) was identified as a potential miR-506-3p target gene with relevance in NAFLD, and this interaction was confirmed via luciferase reporter assay. In the hepatic steatosis model of the present study, miR-506-3p expression level was significantly increased, whereas SIRT1 mRNA/protein levels and AMPK phosphorylation levels were markedly decreased. Transfection of the cells with miR-506-3p mimics led to significant SIRT1 downregulation, while miR-506-3p inhibitor molecules exhibited the opposite effect, with similar trends observed in the phosphorylation status of AMPK. These results suggested that miR-506-3p can inhibit SIRT1 expression and associated AMPK phosphorylation in HepG2 and Huh7 cells in an in vitro hepatic steatosis model system. These data indicated that the miR-506-3p/SIRT1/AMPK axis may be valuable as a therapeutic target in patients affected by NAFLD.

The effects of naringenin and naringin on the glucose uptake and AMPK phosphorylation in high glucose treated HepG2 cells.

BackgroundNaringin and its aglycone naringenin are citrus-derived flavonoids with several pharmacological effects. On the other hand, the mechanism for the anti-diabetic effects of naringenin and naringin are controversial and remain to be clarified further.ObjectiveThis study examined the relationship between glucose uptake and AMP-activated protein kinase (AMPK) phosphorylation by naringenin and naringin in high glucose-treated HepG2 cells.MethodsGlucose uptake was measured using the 2-NBDG fluorescent D-glucose analog. The phosphorylation levels of AMPK and GSK3β (Glycogen synthase kinase 3 beta) were observed by Western blotting. Molecular docking analysis was performed to evaluate the binding affinity of naringenin and naringin to the γ-subunit of AMPK.ResultsThe treatment with naringenin and naringin stimulated glucose uptake regardless of insulin stimulation in high glucose-treated HepG2 cells. Both flavonoids increased glucose uptake by promoting the phosphorylation of AMPK at Thr172 and increased the phosphorylation of GSK3β. Molecular docking analysis showed that both naringenin and naringin bind to the γ-subunit of AMPK with high binding affinities. In particular, naringin showed higher binding affinity than the true modulator, AMP with all three CBS domains (CBS1, 3, and 4) in the γ-subunit of AMPK. Therefore, both naringenin and naringin could be positive modulators of AMPK activation, which enhance glucose uptake regardless of insulin stimulation in high glucose-treated HepG2 cells.ConclusionsThe increased phosphorylation of AMPK at Thr172 by naringenin and naringin might enhance glucose uptake regardless of insulin stimulation in high glucose treated HepG2 cells.

Restoration of the adipogenic gene expression by naringenin and naringin in 3T3-L1 adipocytes

BackgroundNaringenin and its glycoside naringin are well known citrus flavonoids with several therapeutic benefits. Although the anti-adipogenic effects of naringenin and naringin have been reported previously, the detailed mechanism underlying their anti-adipogenesis effects is poorly understood.ObjectivesThis study examined the anti-adipogenic effects of naringenin and naringin by determining differential gene expression patterns in these flavonoids-treated 3T3-L1 adipocytes.MethodsLipid accumulation and triglyceride (TG) content were determined by Oil red O staining and TG assay. Glucose uptake was measured using a 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose fluorescent d-glucose analog. The phosphorylation levels of AMP-activated protein kinase (AMPK) and acetyl Co-A carboxylase (ACC) were observed via Western blot analysis. Differential gene expressions in 3T3-L1 adipocytes were evaluated via RNA sequencing analysis.ResultsNaringenin and naringin inhibited both lipid accumulation and TG content, increased phosphorylation levels of both AMPK and ACC and decreased the expression level of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) in 3T3-L1 adipocytes. RNA sequencing analysis revealed that 32 up-regulated (> 2-fold) and 17 down-regulated (< 0.6-fold) genes related to lipid metabolism, including Acaca, Fasn, Scd1, Mogat1, Dgat, Lipin1, Cpt1a, and Lepr, were normalized to the control level in naringenin-treated adipocytes. In addition, 25 up-regulated (> 2-fold) and 25 down-regulated (< 0.6-fold) genes related to lipid metabolism, including Acaca, Fasn, Fabp5, Scd1, Srebf1, Hmgcs1, Cpt1c, Lepr, and Lrp1, were normalized to the control level by naringin.ConclusionsThe results indicate that naringenin and naringin have anti-adipogenic potentials that are achieved by normalizing the expression levels of lipid metabolism-related genes that were perturbed in differentiated 3T3-L1 cells.

A Bibenzyl Component Moscatilin Mitigates Glycation-Mediated Damages in an SH-SY5Y Cell Model of Neurodegenerative Diseases through AMPK Activation and RAGE/NF-κB Pathway Suppression

Moscatilin can protect rat pheochromocytoma cells against methylglyoxal-induced damage. Elimination of the effect of advanced glycation end-products (AGEs) but activation of AMP-activated protein kinase (AMPK) are the potential therapeutic targets for the neurodegenerative diseases. Our study aimed to clarify AMPK signaling’s role in the beneficial effects of moscatilin on the diabetic/hyperglycemia-associated neurodegenerative disorders. AGEs-induced injury in SH-SY5Y cells was used as an in vitro neurodegenerative model. AGEs stimulation resulted in cellular viability loss and reactive oxygen species production, and mitochondrial membrane potential collapse. It was observed that the cleaved forms of caspase-9, caspase-3, and poly (ADP-ribose) polymerase increased in SH-SY5Y cells following AGEs exposure. AGEs decreased Bcl-2 but increased Bax and p53 expression and nuclear factor kappa-B activation in SH-SY5Y cells. AGEs also attenuated the phosphorylation level of AMPK. These AGEs-induced detrimental effects were ameliorated by moscatilin, which was similar to the actions of metformin. Compound C, an inhibitor of AMPK, abolished the beneficial effects of moscatilin on the regulation of SH-SY5Y cells’ function, indicating the involvement of AMPK. In conclusion, moscatilin offers a promising therapeutic strategy to reduce the neurotoxicity or AMPK dysfunction of AGEs. It provides a potential beneficial effect with AGEs-related neurodegenerative diseases.

LncRNA TUG1 promotes the brown remodeling of white adipose tissue by regulating miR‑204‑targeted SIRT1 in diabetic mice.

The present study aimed to explore the mechanisms of the long non‑coding RNA TUG/miR‑204/SIRT1 axis in the pathogenesis of obesity. For this purpose, a diabetic mouse model was constructed using a high‑fat diet and streptozocin, and the mice were treated with TUG1 virus via tail intravenous injection. Body weight, serum glucose levels, insulin levels and testicular fat, as well as the expression levels of TUG1, miR‑204, Sirtuin 1 (SIRT1) and the AMP‑activated protein kinase (AMPK)/acetyl‑CoA carboxylase (ACC) signaling pathway were detected. Furthermore, the regulatory mechanisms of TUG1/SIRT1 and miR‑204 in the development of diabetes were also explored. The results revealed that the overexpression of TUG1 significantly attenuated body weight, serum glucose levels, insulin tolerance and fatty accumulation in diabetic mice. Furthermore, the overexpression of TUG1 significantly increased the expression SIRT1, adipose triglyceride lipase (ATGL), peroxisome proliferator‑activated receptor α (PPARα), peroxisome proliferator‑activated receptor gamma coactivator 1‑α (PGC‑1α) and uncoupling protein‑1 (UCP‑1), as well as the phosphorylation levels of AMPK and ACC, and decreased the expression of miR‑204 in adipose tissues and 3T3‑L1 cells. miR‑204 inhibitor increased the expression SIRT1, ATGL, PPARα, PGC‑1α and UCP‑1, as well as the phosphorylation levels of AMPK and ACC, and decreased the expression of miR‑204 in the 3T3‑L1 cells; however, the silencing of SIRT1 attenuated these effects. On the whole, the findings of the present study demonstrate that lncRNA TUG1 significantly reverses the development of diabetes by downregulating the expression of miR‑204, and upregulating its targeted SIRT1/AMPK/ACC signaling pathway.

Triptolide induces apoptosis through the calcium/calmodulin‑dependent protein kinase kinaseβ/AMP‑activated protein kinase signaling pathway in non‑small cell lung cancer cells.

Triptolide, a triterpene extracted from the Chinese herb Tripterygiumwilfordii, has been reported to exert multiple bioactivities, including immunosuppressive, anti‑inflammatory and anticancer effects. Although the anticancer effect of triptolide has attracted significant attention, the specific anticancer mechanism in non‑small‑cell lung cancer (NSCLC) remains unclear. The present study aimed to investigate the anticancer effect of triptolide in the H1395 NSCLC cell line and to determine its mechanism of action. The results revealed that triptolide significantly inhibited the cell viability of NSCLC cells in a dose‑dependent manner, which was suggested to be through inducing apoptosis. In addition, triptolide was revealed to activate the calcium (Ca2+)/calmodulin‑dependent protein kinase kinaseβ (CaMKKβ)/AMP‑activated protein kinase (AMPK) signaling pathway by regulating the intracellular Ca2+ concentration levels, which increased the phosphorylation levels of AMPK and reduced the phosphorylation levels of AKT, ultimately leading to apoptosis. The CaMKKβ blocker STO‑609 and the AMPK blocker Compound C significantly inhibited the apoptosis‑promoting effect of triptolide. In conclusion, the results of the present study suggested that triptolide may induce apoptosis through the CaMKKβ‑AMPK signaling pathway and may be a promising drug for the treatment of NSCLC.

Phloretin Modulates Human Th17/Treg Cell Differentiation In Vitro via AMPK Signaling.

Objective We conducted studies to explore the effect of phloretin on glucose uptake, proliferation, and differentiation of human peripheral blood CD4+ T cells and investigated the mechanism of phloretin on inducing Th17/Treg development. Methods Naïve CD4+ T cells were purified from peripheral blood of healthy volunteers, stimulated with anti-CD3/CD28 antibodies, and polarized in vitro to generate Th17 or Treg cells. Glucose uptake, proliferation, cell cycle, protein expression (phospho-Stat3, phospho-Stat5), and Th17 and Treg cell numbers were analyzed by flow cytometry. AMP-activated protein kinase (AMPK) signaling was analyzed by western blot. Results and Discussion. Phloretin could inhibit the glucose uptake and proliferation of activated CD4+ T cells. The proliferation inhibition was due to the G0/G1 phase arrest. Phloretin decreased Th17 cell generation and phospho-Stat3 expression as well as increased Treg cell generation and phospho-Stat5 expression in the process of inducing Th17/Treg differentiation. The phosphorylation level of AMPK was significantly enhanced, while the phosphorylation level of mTOR was significantly decreased in activated CD4+ T cells under phloretin treatment. The AMPK signaling inhibitor compound C (Com C) could neutralize the effect of phloretin, while the agonist 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) could impact the Th17/Treg balance similar to phloretin during Th17/Treg induction. Conclusion Our results suggest that phloretin can mediate the Th17/Treg balance by regulating metabolism via the AMPK signal pathway.

ω-hydroxyundec-9-enoic acid induction of breast cancer cells apoptosis through generation of mitochondrial ROS and phosphorylation of AMPK.

This study was performed to evaluate the anticancer effect of ω-hydroxyundec-9-enoic acid (ω-HUA), a microbial bio-catalyst product in breast cancer cells, through AMP-activated protein kinase (AMPK) regulation. ω-HUA mediated apoptosis was induced in breast cancer cells by AMPK activation, loss of mitochondrial membrane potential, and reactive oxygen species (ROS) generation. ω-HUA treatment of breast cancer cells increased the AMPK phosphorylation levels, cleaved caspase-3, and poly (ADP-ribose) polymerase (PARP) proteins. In addition, anti-apoptotic members, such as Bcl-2, were downregulated, while Bax, a pro-apoptotic member, was upregulated. ω-HUA decreased the mitochondrial membrane potential while increasing the expression of cytochrome c (cyt c). Treating the cells with compound C, an AMPK inhibitor, reversed the phenomena, leading to an increase in cell viability and a decrease in apoptosis induction. Treating the cells with an ROS scavenger, N-acetyl cysteine (NAC), led to AMPK inactivation and apoptosis inhibition, allowing the recovery of cell health. In conclusion, ω-HUA sequentially caused the production of mitochondrial ROS and the consequent AMPK activation, thereby inducing apoptosis in breast cancer cells. Thus, ω-HUA may prove useful as an anticancer agent that targets AMPK in breast cancer cells.

Adiponectin Reduces Embryonic Loss Rate and Ameliorates Trophoblast Apoptosis in Early Pregnancy of Mice with Polycystic Ovary Syndrome by Affecting the AMPK/PI3K/Akt/FoxO3a Signaling Pathway

Reports in recent years have suggested that adiponectin (APN) improves insulin resistance and inhibits apoptosis by activating the AMP-activated protein kinase (AMPK) pathway and the PI3K/Akt signaling pathway after binding to its receptor. This study aims to explore the mechanism by which APN reduces embryo loss rate and trophoblast apoptosis in early pregnancy of mice with polycystic ovary syndrome (PCOS). PCOS mice were subcutaneously injected with APN (10 μg mg kg−1 day−1) on 11 consecutive days from the 3rd day of pregnancy onwards to observe the change of the embryo loss rate of PCOS mice induced by APN. Quantitative real-time PCR and Western blot were used to determine the relative expressions of mRNA and the proteins AMPK, PI3K, and Akt in mouse uterine tissue. At the same time, primary cultured mouse villous trophoblast cells were used to further explore the underlying mechanisms in vitro. APN significantly reduces the pregnancy loss rate of PCOS mice. At the same time, APN increases phosphorylation and mRNA expression levels of AMPK, PI3K, and Akt in PCOS mouse uterine tissue. In addition, trophoblast cells of model mice were treated with APN and inhibitors, and APN was found to reduce trophoblast cell apoptosis by affecting the phosphorylation levels of AMPK, PI3K, Akt, and FoxO3a proteins. APN reduces the embryo loss rate and ameliorates trophoblast apoptosis in PCOS mice by affecting the AMPK/PI3K/AKT/FoxO3a signaling pathway.