AMPK Phosphorylation Research Articles

Branched-chain amino acids alter cellular redox to induce lipid oxidation and reduce de novo lipogenesis in the liver.

Metabolic and molecular interactions between branched-chain amino acid (BCAA) and lipid metabolism are evident in insulin-resistant tissues. However, it remains unclear whether insulin resistance is a prerequisite for these relationships and whether BCAAs or their metabolic intermediates can modulate hepatic lipid oxidation and synthesis. We hypothesized that BCAAs can alter hepatic oxidative function and de novo lipogenesis, independent of them being anaplerotic substrates for the mitochondria. Mice (C57BL/6NJ) were reared on a low-fat (LF), LF diet plus 1.5X BCAAs (LB), high-fat (HF) or HF diet plus 1.5X BCAAs (HB) for 12 wk. Hepatic metabolism was profiled utilizing stable isotopes coupled to mass spectrometry and nuclear magnetic resonance, together with fed-to-fasted changes in gene and protein expression. A greater induction of lipid oxidation and ketogenesis on fasting was evident in the BCAA-supplemented, insulin-sensitive livers from LB mice, whereas their rates of hepatic de novo lipogenesis remained lower than their LF counterparts. Onset of insulin resistance in HF and HB mice livers blunted these responses. Whole body turnover of BCAAs and their ketoacids, their serum concentrations, and the ketogenic flux from BCAA catabolism, all remained similar between fasted LF and LB mice. This suggested that the impact of BCAAs on lipid metabolism can occur independent of them or their degradation products fueling anaplerosis through the liver mitochondria. Furthermore, the greater induction of lipid oxidation in the LB livers accompanied higher mitochondrial NADH/NAD+ ratio and higher fed-to-fasting phosphorylation of AMPKα and ACC. Taken together, our results provide evidence that BCAA supplementation, under conditions of insulin sensitivity, improved the feeding-to-fasting induction of hepatic lipid oxidation through changes in cellular redox, thus providing a favorable biochemical environment for flux through β-oxidation and lower de novo lipogenesis.NEW & NOTEWORTHY Branched-chain amino acids (BCAAs) have been shown to modulate lipid metabolic networks in various tissues, especially during insulin resistance. In this study we show that the dietary supplementation of BCAAs to normal, insulin-sensitive mice resulted in higher mitochondrial NADH:NAD+ ratios and AMPK activation in the liver. This change in the cellular redox status provided an optimal metabolic milieu to increase fatty acid oxidation while keeping the rates of de novo lipogenesis lower in the BCAA-supplemented mice livers.

Quercetin-3-O-rutinoside from Moringa oleifera Downregulates Adipogenesis and Lipid Accumulation and Improves Glucose Uptake by Activation of AMPK/Glut-4 in 3T3-L1 Cells.

Natural product-based therapeutic alternatives have drawn immense interest to deal with growing incidence of metabolic disorders. Rutin (quercetin-3-O-rutinoside) is found in a variety of fruits, vegetables, and plant beverages. In the present study, rutin was isolated from Moringa oleifera Lam., leaves and its anti-lipidemic and anti-adipogenic activity was evaluated through inhibition of key digestive enzymes and in vitro cell culture experiments using 3T3-L1 adipocytes. Rutin treatment substantially reduced α-glucosidase and pancreatic lipase activities with IC50 values of 40 and 35 μg/ml, respectively. MTT assay with 3T3-L1 cells demonstrated the non-toxic effect of rutin up to 160 μg/ml. Oil Red O-stained images of rutin-treated 3T3-L1 cells depicted that rutin considerably reduced lipid content and adipogenesis (79.9%), and enhanced glycerol release in 3T3-L1 cells when compared to untreated cells. Rutin significantly (p < 0.05) enhanced glucose uptake in 3T3-L1 adipocytes and also led to reduced levels of leptin but enhanced levels of adiponectin. Western blot analysis of rutin-treated (40 µg/ml) adipocytes showed phosphorylation of AMPK, upregulated expression of Glut-4 (1.31-fold) and UCP-1 (1.47-fold), but downregulated expression of PPAR-γ by 0.73-fold. At transcriptional level, similar trends were observed in the mRNA expression of the above genes, except AMPK. Our results demonstrate that rutin isolated from M. oleifera significantly alleviates lipid content and adipogenesis, and improves glucose uptake through regulating PPAR-γ and AMPK signaling pathways; thus, rutin can be considered as a potential therapeutic agent against adiposity and glucose intolerance.

Combined chronic copper exposure and aging lead to neurotoxicity in vivo

The environment, containing pollutants, toxins, and transition metals (copper, iron, manganese, and zinc), plays a critical role in neurodegenerative disease development. Copper occupational exposure increases Parkinson’s disease (PD) risk. Previously, we determined the mechanisms by which copper induces dopaminergic cell death in vitro. The copper transporter protein 1 (Ctr1) overexpression led to intracellular glutathione depletion potentiating caspase-3 mediated cell death; oxidative stress was primarily cytosolic, and Nrf2 was upregulated mediating an antioxidant response; and protein ubiquitination, AMPK-Ulk1 signaling, p62, and Atg5-dependent autophagy were increased as a protective mechanism. However, the effect of chronic copper exposure on the neurodegenerative process has not been explored in vivo. We aimed to elucidate whether prolonged copper treatment reproduces PD features and mechanisms during aging. Throughout 40 weeks, C57BL/6J male mice were treated with copper at 0, 100, 250, and 500 ppm in the drinking water. Chronic copper exposure altered motor function and induced dopaminergic neuronal loss, astrocytosis, and microgliosis in a dose-dependent manner. α-Synuclein accumulation and aggregation were increased in response to copper, and the proteasome and autophagy alterations, previously observed in vitro, were confirmed in vivo, where protein ubiquitination, AMPK phosphorylation, and the autophagy marker LC3-II were also increased by copper exposure. Finally, nitrosative stress was induced by copper in a concentration-dependent fashion, as evidenced by increased protein nitration. To our knowledge, this is the first study combining chronic copper exposure and aging, which may represent an in vivo model of non-genetic PD and help to assess potential prophylactic and therapeutic approaches. Data AvailabilityThe data underlying this article are available in the article.

Carnosol inhibits cerebral ischemia-reperfusion injury by promoting AMPK activation

BackgroundCarnosol is a phytopolyphenol (diterpene) found and extracted from plants of Mediterranean diet, which has anti-tumor, anti-inflammatory and antioxidant effects. However, its role in ischemic stroke has not been elucidated. MethodsPrimary neurons subjected to oxygen-glucose deprivation (OGD) was used to investigate the effect of carnosol in vitro. A mouse MCAO model was used to evaluate the effect of carnosol on ischemic stroke in vivo. The mRNA level of inflammatory and apoptosis-related genes was determined by RT-PCR. The protein level of total and phosphorylated AMPK was determined by WB. H&E and Immunofluorescent assay was used to investigate the necrosis, inflammation and apoptosis in brain tissue. ResultsCarnosol protected the activity of primary neurons subjected to oxygen-glucose deprivation (OGD) in vitro, as well as inhibited inflammation and apoptosis. Furthermore, carnosol could significantly reduce the infarct and edema volume and protect against neurological deficit in vivo, and had a significant inhibitory effect on brain neuroinflammation and apoptosis. Mechanically, carnosol could activate AMPK, and the effect of carnosol on cerebral ischemia-reperfusion injury cell model could be abolished by AMPK phosphorylation inhibitor. ConclusionCarnosol has a protective effect on ischemic stroke, and this effect is achieved through AMPK activation. Our study demonstrates the protective effect of carnosol on cerebral ischemia-reperfusion injury and provides a new perspective for the clinical treatment of ischemic stroke.

Ginkgo biloba extract protects against diabetic cardiomyopathy by restoring autophagy via adenosine monophosphate-activated protein kinase/mammalian target of the rapamycin pathway modulation.

Studies demonstrated that Ginkgo biloba extract (GBE) played a cardioprotective role in diabetic conditions. Impaired autophagy is one of the mechanisms underlying diabetic cardiomyopathy (DCM). The effect of GBE on autophagy has been observed in several diseases; however, whether GBE can ameliorate DCM by regulating autophagy remains unclear. Here, we investigated the effect of GBE on DCM and the potential mechanisms regarding autophagy using a streptozotocin (STZ)-induced diabetic rat model and a high-glucose (HG)-stimulated H9C2 cell model. We demonstrated that GBE attenuated metabolic disturbances, improved cardiac function, and reduced myocardial pathological changes in diabetic rats. Impaired autophagy as well as dysregulation of the adenosine monophosphate-activated protein kinase/ mammalian target of the rapamycin (AMPK/mTOR) signaling pathway were observed in diabetic hearts, as evidenced by the reduced conversion of LC3B-I to LC3B-II along with excessive p62 accumulation, decreased AMPK phosphorylation, and increased mTOR phosphorylation, which could be reversed by GBE treatment. In vitro, GBE reduced the apoptosis induced by HG in H9C2 cells by activating AMPK and inhibiting mTOR to restore autophagy. However, this effect was inhibited by the AMPK inhibitor Compound C. In conclusion, the ameliorative effect of GBE on DCM might be dependent on the restoration of autophagy through modulation of the AMPK/mTOR pathway.

Kazinol B protects H9c2 cardiomyocytes from hypoxia/reoxygenation-induced cardiac injury by modulating the AKT/AMPK/Nrf2 signalling pathway

Context Kazinol B (KB), an isoprenylated flavan derived from Broussonetia kazinoki Sieb. (Moraceae) root, has long been used in folk medicine. Objective This study examines the protective effects of KB and its underlying mechanisms in hypoxia and reoxygenation (H/R)-induced cardiac injury in H9c2 rat cardiac myoblasts. Materials and methods H9c2 cells were incubated with various concentrations of KB (0, 0.3, 1, 3, 10 and 30 μM) for 2 h and then subjected to H/R insults. The protective effects of KB and its underlying mechanisms were explored. Results KB significantly elevated cell viability (1 μM, 1.21-fold; 3 μM, 1.36-fold, and 10 μM, 1.47-fold) and suppressed LDH release (1 μM, 0.77-fold; 3 μM, 0.68-fold, and 10 μM, 0.59-fold) in H/R-induced H9c2 cells. Further, 10 μM KB blocked apoptotic cascades, as shown by the Annexin-V/PI (0.41-fold), DNA fragmentation (0.51-fold), caspase-3 (0.52-fold), PARP activation (0.27-fold) and Bax/Bcl-2 expression (0.28-fold) assays. KB (10 μM) downregulated reactive oxygen species production (0.51-fold) and lipid peroxidation (0.48-fold); it upregulated the activities of GSH-Px (2.08-fold) and SOD (1.72-fold). KB (10 μM) induced Nrf2 nuclear accumulation (1.94-fold) and increased ARE promoter activity (2.15-fold), HO-1 expression (3.07-fold), AKT (3.07-fold) and AMPK (3.07-fold) phosphorylation. Nrf2 knockdown via using Nrf2 siRNA abrogated KB-mediated protective effects against H/R insults. Moreover, pharmacological inhibitors of AKT and AMPK also abrogated KB-induced Nrf2 activation and its protective function. Discussion and conclusions KB prevented H/R-induced cardiomyocyte injury via modulating the AKT and AMPK-mediated Nrf2 induction. KB might be a promising drug candidate for managing ischemic cardiac disorders.

The Anti-Aging Hormone Klotho Promotes Retinal Pigment Epithelium Cell Viability and Metabolism by Activating the AMPK/PGC-1α Pathway

Initially discovered by Makuto Kuro-o in 1997, Klotho is a putative aging-suppressor gene when overexpressed and accelerates aging when deleted in mice. Previously, we showed that α-Klotho regulates retinal pigment epithelium (RPE) functions and protects against oxidative stress. However, the mechanisms by which Klotho influences RPE and retinal homeostasis remain elusive. Here, by performing a series of in vitro and in vivo experiments, we demonstrate that Klotho regulates cell viability under oxidative stress, mitochondrial gene expression and activity by inducing the phosphorylation of AMPK and p38MAPK, which in turn phosphorylate and activate CREB and ATF2, respectively, triggering PGC-1α transcription. The inhibition of Klotho in human RPE cells using CRISPR-Cas9 gene editing confirmed that a lack of Klotho negatively affects RPE functions, including mitochondrial activity and cell viability. Proteomic analyses showed that myelin sheath and mitochondrial-related proteins are downregulated in the RPE/retina of Kl-/- compared to WT mice, further supporting our biochemical observations. We conclude that Klotho acts upstream of the AMPK/PGC-1α pathway and regulates RPE/retinal resistance to oxidative stress, mitochondrial function, and gene and protein expressions. Thus, KL decline during aging could negatively impact retinal health, inducing age-related retinal degeneration.

Sitagliptin Ameliorates Creb5/lncRNA ENSMUST00000213271-Mediated Vascular Endothelial Dysfunction in Obese Mice.

Obesity is mediated by the changes in dyslipidemia, oxidative stress, and inflammation, leading to vascular endothelial dysfunction. Glucagon-like peptide-1 (GLP-1) analogues and dipeptidyl peptidase-4 inhibitors prevent the development of endothelial dysfunction. However, the underlying mechanism still remains largely unclear. Long non-coding RNAs (lncRNAs), one class of non-coding small RNAs, have been shown to exert a regulatory impact on the endothelial function in obesity. This study aimed to investigate whether the elevation of GLP-1 by a DPP-4 inhibitor sitagliptin improved vascular endothelial function by modulating lncRNAs in obese mice and to clarify the underlying molecular mechanism. Male C57BL/6J mice were fed a high-fat diet for 4 months to induce obesity and some obese mice were treated with sitagliptin for the last 1 month. Levels of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and glucagon-like peptide-1 (GLP-1) in plasma were detected by ELISA. LncRNA expression profile was analyzed via microarray. Aortic relaxations were examined by myograph. Protein expressions and phosphorylations were determined using western blot. The differentially expressed lncRNAs were validated using qRT-PCR. Obese mice exhibited increased levels of TC and LDL, decreased concentrations of HDL and GLP-1 in plasma, and impaired aortic endothelium-dependent relaxations; such effects could be reversed by sitagliptin. Moreover, the altered expression profile of lncRNAs in the obese mouse aortae could be modulated by sitagliptin. Consistent with microarray analysis, qRT-PCR also revealed that lncRNA ENSMUST00000213271 was up-regulated in obese mouse aortae and aortic endothelial cells (ECs), which could be down-regulated by sitagliptin. Creb5 silencing reduced lncRNA ENSMUST00000213271 in obese mouse ECs. Knockdown of either Creb5 or lncRNA ENSMUST00000213271 restored the activation of AMPK/eNOS in obese mouse ECs. Furthermore, sitagliptin also suppressed Creb5 and lncRNA ENSMUST00000213271 and increased the phosphorylations of AMPK and eNOS in obese mice. Creb5/lncRNA ENSMUST00000213271 mediated vascular endothelial dysfunction through inhibiting AMPK/eNOS cascade in obesity. Elevation of GLP-1 by sitagliptin possibly improved endothelial function by suppressing Creb5/lncRNA ENSMUST00000213271 and subsequently restoring AMPK/eNOS activation in obese mice. This study will provide new evidence for the benefits of GLP-1 against vasculopathy in obesity.

Jiedu Tongluo Baoshen formula enhances renal tubular epithelial cell autophagy to prevent renal fibrosis by activating SIRT1/LKB1/AMPK pathway

Renal fibrosis, an important pathological change in the development of diabetic kidney disease (DKD), urgently needs new treatment methods clinically. The Jiedu Tongluo Baoshen (JTBF) formula was created based on the theory of toxic damage to the kidney collaterals, and a variety of active ingredients in JTBF have inhibitory effects on epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM). In this study, the Ultra Performance Liquid Chromatography (UPLC) was employed to analyze the effective ingredients in the JTBF formula. After screening in the PubChem database, we identified 94 active compounds of JTBF and predicted the SIRT1 pathway as potential targets through network pharmacology. In addition, in the high fat diet (HFD)+Streptozocin (STZ)-induced DKD rat model and high glucose (HG)-induced NRK-52E cell model, JTBF treatment activates the phosphorylation of LKB1 and AMPK and enhances the autophagy activity of NRK-52E cells, thereby reducing the accumulation of EMT and ECM. These results have been confirmed in vivo and in vitro experiments. JTBF enhances the autophagy activity of renal tubular epithelial cells and inhibits the progression of DKD renal fibrosis by activating the SIRT1/LKB1/AMPK signal pathway. This study provides new insights into the molecular mechanism of JTBF to prevent and treat DKD renal fibrosis.

Rho-kinase inhibitor restores glomerular fatty acid metabolism in diabetic kidney disease

The small GTPase Rho and its effector Rho-kinase (ROCK) are activated in the diabetic kidney, and recent studies decade have demonstrated that ROCK signaling is an integral pathway in the progression of diabetic kidney disease. We previously identified the distinct role of ROCK1, an isoform of ROCK, in fatty acid metabolism in diabetic glomeruli. However, the effect of pharmacological intervention for ROCK1 is not clear. In the present study, we show that the inhibition of ROCK1 by Y-27632 and fasudil restores fatty acid oxidation in the glomeruli. Mechanistically, these compounds optimize fatty acid utilization and redox balance in mesangial cells via AMPK phosphorylation and the subsequent induction of PGC-1α. A further in vivo study showed that the inhibition of ROCK1 suppressed the downregulation of the fatty acid oxidation-related gene expression in glomeruli and mitochondrial fragmentation in the mesangial cells of db/db mice. These observations indicate that ROCK1 could be a promising therapeutic target for diabetic kidney disease through a mechanism that improves glomerular fatty acid metabolism.

Exposure to dipentyl phthalate in utero disrupts the adrenal cortex function of adult male rats by inhibiting SIRT1/PGC-1α and inducing AMPK phosphorylation.

Di-n-pentyl phthalate (DPeP) is an endocrine-disrupting phthalate plasticizer. The objective of this study was to investigate the effect of DPeP on adrenocortical function in adult male rats following in utero exposure. DPeP (0, 10, 50, 100, and 500 mg/kg/day) was administered by gavage to pregnant Sprague-Dawley rats from gestational day 14 to 21. The morphology and function of the adrenal cortex in 56-day-old male offspring were studied. DPeP at 100 and 500 mg/kg/day significantly reduced serum aldosterone levels and at 500 mg/kg/day markedly reduced corticosterone and adrenocorticotropic hormone levels. DPeP at 10-500 mg/kg markedly reduced the thickness of zona glomerulosa without affecting the thickness of zona fasciculata. DPeP significantly downregulated the expression of Agtr1a, Mc2r, Scarb1, Cyp11a1, Hsd3b1, Cyp21, Cyp11b1, Cyp11b2, Nr5a1, Nr4a2, and Bcl2 genes as well as their proteins. DPeP at 500 mg/kg/day significantly increased phosphorylated AMPK, while DPeP at 100 mg/kg/day and higher doses reduced phosphorylated AKT1 and total SIRT1 level. DPeP at 100 and 500 μM markedly induced reactive oxygen species and apoptosis in H295R cells after 24 h of culture. In conclusion, in utero exposure to DPeP disrupts adrenocortical function of the adult male offspring by (1) increasing AMPK phosphorylation and decreasing AKT1 phosphorylation and SIRT1 levels, (2) reducing adrenocorticotropic hormone levels, and (3) possibly inducing oxidative stress and apoptosis.

Rapid metabolic reprogramming mediated by the AMP-activated protein kinase during the lytic cycle of Toxoplasma gondii

The ubiquitous pathogen Toxoplasma gondii has a complex lifestyle with different metabolic activities at different stages that are intimately linked to the parasitic environments. Here we identified the eukaryotic regulator of cellular homeostasis AMP-activated protein kinase (AMPK) in Toxoplasma and discovered its role in metabolic programming during parasite’s lytic cycle. The catalytic subunit AMPKα is quickly phosphorylated after the release of intracellular parasites to extracellular environments, driving energy-producing catabolism to power parasite motility and invasion into host cells. Once inside host cells, AMPKα phosphorylation is reduced to basal level to promote a balance between energy production and biomass synthesis, allowing robust parasite replication. AMPKγ depletion abolishes AMPKα phosphorylation and suppresses parasite growth, which can be partially rescued by overexpressing wildtype AMPKα but not the phosphorylation mutants. Thus, through the cyclic reprogramming by AMPK, the parasites’ metabolic needs at each stage are satisfied and the lytic cycle progresses robustly.

Ellagic acid induces apoptosis and autophagy in colon cancer through the AMPK/mTOR pathway

Ellagic acid (EA), found in fruits and foods, has been shown to be effective in the treatment of breast, colon and bladder cancer. However, due to the complexity of colon cancer, the therapeutic mechanism of EA for colon cancer is still unclear. Cell Counting Kit-8 (CCK-8) assay were employed to investigate the cell proliferation. Western blotting and flow cytometry assays were utilized to investigate apoptosis and autophagy in CRC cells (HCT116), respectively. Moreover, western blotting and luciferase reporter assays were evaluated the effect of EA on AMPK/mTOR pathway. Through flow cytometry analysis, EA could promote the apoptosis of HCT116 cells. In addition, EA can reduce the phosphorylation of mTOR, promoted phosphorylation of AMPK, and induced autophagy in HCT116 cells. Also, Dorsomorphin pretreatment can reduce the expression of autophagy protein, which indicates that EA induces autophagy through AMPK/mTOR pathway. These results suggest that EA inhibits the growth of colon cancer through AMPK/mTOR pathway and induces apoptosis and protective autophagy.

Corilagin alleviates intestinal ischemia/reperfusion injury by relieving oxidative stress and apoptosis via AMPK/Sirt1-autophagy pathway.

Intestinal ischemia/reperfusion (II/R) injury is a common pathological process with high clinical morbidity and mortality. Autophagy plays an important role in the pathological development of II/R. Corilagin (CA) is a natural ellagitannin with various pharmacological effects such as autophagy regulation, antioxidant, and antiapoptosis. However, whether CA alleviates II/R injury is still unclear. In this study, we had found that CA significantly attenuated II/R induced intestinal tissue pathological damage, oxidative stress, and cell apoptosis in rats. Further studies showed that CA significantly promoted AMPK phosphorylation and sirt1 expression, and thus activated autophagy by upregulating protein expression of autophagy-related proteins Beclin1 and LC3II and promoting SQSTM1/P62 degradation both in vivo and in vitro. Inhibition of AMPK phosphorylation by its inhibitor compound C(CC) significantly abolished CA-mediated autophagy activation and the relievable effects on oxidative stress and apoptosis in vitro, suggesting the excellent protective activity of CA against II/R injury via AMPK/Sirt1-autophagy pathway. These findings confirmed the potent effects of CA against II/R injury, and provided novel insights into the mechanisms of the compound as a potential candidate for the treatment of II/R.

Reduced Expression Level of Protein Phosphatase PPM1E Serves to Maintain Insulin Secretion in Type 2 Diabetes.

Reversible phosphorylation is an important regulatory mechanism. Regulation of protein phosphorylation in β-cells has been extensively investigated, but less is known about protein dephosphorylation. To understand the role of protein dephosphorylation in β-cells and type 2 diabetes (T2D), we first examined mRNA expression of type 2C family (PP2C) of protein phosphatases in islets from T2D donors. Phosphatase expression overall was changed in T2D, and that of PPM1E was most markedly downregulated. PPM1E expression correlated inversely with HbA1c. Silencing of PPM1E increased glucose-stimulated insulin secretion (GSIS) in INS-1 832/13 cells and/or islets from T2D patients while PPM1E overexpression decreased GSIS. Increased GSIS following PPM1E silencing was associated with decreased oxidative stress, elevated cytosolic Ca2+ levels and ATP/ADP ratio, increased hyperpolarization of the inner mitochondrial membrane and phosphorylation of CaMKII, AMPK and acetyl-coA carboxylase. Silencing of PPM1E, however, did not change insulin content. Increased GSIS, cell viability, and activation of AMPK upon metformin treatment in β-cells were observed upon PPM1E silencing. Thus, protein dephosphorylation via PPM1E abrogates GSIS. Consequently, reduced PPM1E expression in T2D may be a compensatory response of β-cells to uphold insulin secretion under metabolic duress. Targeting PPM1E in β-cells may thus represent a novel therapeutic strategy for treatment of T2D.

Baicalein limits osteoarthritis development by inhibiting chondrocyte ferroptosis

Osteoarthritis (OA) is a common degenerative disease of articular cartilage, and there is currently no effective treatment. Baicalein, a flavonoid extracted from plants of the Scutellaria genus, has frequently been used in the past as an anti-inflammatory and anti-allergic therapy. This study investigated the effect of baicalein on OA development. For in vivo study, a destabilization of the medial meniscus (DMM)-induced OA model was established in 8-week-old wild-type (WT) and AMPKα-knockout (KO) mice, while an in vitro study was performed using chondrocytes in an OA microenvironment induced by interleukin-1β (IL-1β) exposure. We found that baicalein alleviated OA development in vivo and exerted a chondroprotective effect in vitro by suppressing chondrocyte ferroptosis. Baicalein reduced OA-related pain sensitivity by inhibiting ferroptosis of chondrocytes in OA mice. Baicalein also facilitated AMPK holoenzyme assembly, stability, and activity and suppressed ferroptosis by inducing AMPKα phosphorylation in chondrocyte. In addition, AMPKα preserved nuclear factor erythroid 2-related factor 2(Nrf2) abundance in chondrocytes and induced Nrf2 into nucleus by promoting Keap1 degradation. Meanwhile, Nrf2 increased expression of heme oxygenase-1(HO-1) to inhibit chondrocyte lipid ROS. Taken together, these results showed that baicalein alleviated OA development by improving the activity of AMPK/Nrf2/HO-1 signaling to inhibit chondrocyte ferroptosis, revealing baicalein to be a potential therapeutic strategy for OA.

Ginsenoside Rd promotes omentin secretion in adipose through TBK1-AMPK to improve mitochondrial biogenesis via WNT5A/Ca2+ pathways in heart failure

Ginsenoside Rd is an active ingredient in Panax ginseng CA Mey and can be absorbed into the adipose tissue. Adipokines play an important role in the treatment of cardiovascular diseases. However, the potential benefit of Rd on heart failure (HF) and the underlying mechanism associated with the crosstalk between adipocytes and cardiomyocytes remains to be illustrated. Here, the results identified that Rd improved cardiac function and inhibited cardiac pathological changes in transverse aortic constriction (TAC), coronary ligation (CAL) and isoproterenol (ISO)-induced HF mice. And Rd promoted the release of omentin from the adipose tissue and up-regulated omentin expression in lipopolysaccharide (LPS)-induced 3T3-L1 adipocytes. Further, Rd could increase TBK1 and AMPK phosphorylation in adipocytes. And also, the TBK1-AMPK signaling pathway regulated the expression of omentin in LPS-induced adipocytes. Moreover, the omentin mRNA expression was significantly decreased by TBK1 knockdown in LPS-induced 3T3-L1 adipocytes. Additionally, molecular docking and SPR analysis confirmed that Rd had a certain binding ability with TBK1, and co-treatment with TBK1 inhibitors or TBK1 knockdown partially abolished the effect of Rd on increasing the omentin expression and the ratio of p-AMPK to AMPK in adipocytes. Moreover, we found that circulating omentin level diminished in the HF patients compared with healthy subjects. Meanwhile, the adipose tissue-specific overexpression of omentin improved cardiac function, reduced myocardial infarct size and ameliorated cardiac pathological features in CAL-induced HF mice. Consistently, exogenous omentin reduced mtROS levels and restored ΔψM to improve oxygen and glucose deprivation (OGD)-induced cardiomyocytes injury. Further, omentin inhibited the WNT5A/Ca2+ signaling pathway and promoted mitochondrial biogenesis function to ameliorate myocardial ischemia injury. However, WNT5A knockdown inhibited the impairment of mitochondrial biogenesis and partially counteracted the cardioprotective effect of omentin in vitro. Therefore, this study indicated that Rd promoted omentin secretion from adipocytes through the TBK1-AMPK pathway to improve mitochondrial biogenesis function via WNT5A/Ca2+ signaling pathway to ameliorate myocardial ischemia injury, which provided a new therapeutic mechanism and potential drugs for the treatment of HF.

Undaria pinnatifida (Wakame) Intake Ameliorates High-Fat Diet-Induced Glucose Intolerance via Promoting GLUT4 Expression and Membrane Translocation in Muscle.

Type 2 diabetes mellitus (T2DM), a lifestyle-related disease, is developed due to eating habits and decreased physical activity. Diabetes also increases the risk of cancer and major neurodegenerative diseases; controlling the onset of diabetes helps prevent various illnesses. Eating seaweed, such as Undaria pinnatifida (wakame), is a part of the Asian food culture. Therefore, we analyzed the antidiabetic effect of wakame intake using the high-fat diet-induced diabetes mouse model. Furthermore, we analyzed the effect of wakame extract on the cell membrane translocation of glucose transporter-4 (GLUT4) and activation of insulin signal molecules, such as AKT and AMPK, in insulin-sensitive tissues. Differentiated C2C12 cells were incubated with wakame components. The membrane translocation of GLUT4 and phosphorylation of AKT and AMPK were investigated with immunofluorescence staining and Western blotting, respectively. Also, male C57BL/6J mice were fed the normal diet (ND), high-fat diet (HFD), ND with 1% wakame powder (ND + W), or HFD with 1% wakame powder (HFD + W). We evaluated the effect of wakame intake on high-fat diet-induced glucose intolerance using an oral glucose tolerance test. Moreover, we analyzed insulin signaling molecules, such as GLUT4, AKT, and AMPK, in muscle using Western blotting. GLUT4 membrane translocation was promoted by wakame components. Also, GLUT4 levels and AKT and AMPK phosphorylation were significantly elevated by wakame components in C2C12 cells. In addition, the area under the curve (AUC) of the HFD + W group was significantly smaller than that of the HFD group. Furthermore, the level of GLUT4 in the muscle was increased in the wakame intake group. This study revealed that various wakame components exerted antidiabetic effects on the mice on a high-fat diet by promoting glucose uptake in the skeletal muscle, enhancing GLUT4 levels, and activating AKT and AMPK.

Autocrine role of senescent cardiac fibroblasts-derived extracellular vesicles.

Cellular senescence is a highly stable state associated with cell cycle arrest, that is elicited in response to various stresses. The accumulation of senescent cells in tissues drives age-related diseases. Recent studies have shown that the cellular senescence enhances an extracellular vesicles (EV) secretion. EV are lipid-bilayer-capsuled particles released by various cells mediating cell-to-cell communication. It was recently reported that EV secreted by the senescent cells had several functions such as cancer cell proliferation and immune cell activation. In the present study, we investigated whether senescent cardiac fibroblasts-derived EV play an autocrine/paracrine role in the heart cells. Neonatal rat cardiac fibroblasts (NRCFs) were treated with doxorubicin (DOX) to induce cellular senescence. EV were isolated from NRCFs culture media. The vehicle-treated NRCFs-derived EV (D0-EV, 72 hr) increased a living cell number in NRCFs, which was attenuated by DOX (1,000 nM)-treated NRCFs-derived EV (D103-EV, 72 hr). While D0-EV did not affect protein concentration in NRCFs, D103-EV decreased it. Furthermore, D103-EV significantly increased a ratio of microtubule-associated protein 1 light chain 3 (LC3)-II to LC3-I in NRCFs, indicating an induction of autophagy. In addition, D103-EV increased phosphorylation of adenosine monophosphate-activated kinase (AMPK) α in NRCFs. In neonatal rat cardiomyocytes, however, NRCFs-derived EV (72 hr) had no effect on the living cell number, protein concentration, and ratio of LC3-II to LC3-I. In conclusion, we for the first time revealed that DOX-induced senescent NRCFs-derived EV induce autophagy in NRCFs perhaps partly through the activation of AMPKα.

Chemerin affects the expression of angiogenesis-related factors in the porcine endometrium during early pregnancy and the oestrous cycle: an in vitro study.

The appropriate course of angiogenesis in the endometrium is crucial for pregnancy establishment and maintenance. Very little is known about the factors linking vessel formation and immune system functioning. We hypothesised that chemerin, an adipokine known for its involvement in the regulation of energy balance and immunological functions, may act as a potent regulator of endometrial angiogenesis during early pregnancy in pigs. Porcine endometrial tissue explants were obtained from pregnant pigs on days 10-11, 12-13, 15-16 and 27-28, and on days 10-12 of the oestrous cycle. The explants were in vitro cultured for 24h in the presence of chemerin (100, 200ng/mL) or in medium alone (control). We evaluated the in vitro effect of chemerin on the secretion of vascular endothelial growth factors A-D (VEGF-A-D), placental growth factor (PlGF), basic fibroblast growth factor (bFGF) and angiopoietin 1 and 2 (ANG-1, ANG-2) with the ELISA method. The protein abundance of angiogenesis-related factor receptors, VEGF receptors 1-3 (VEGFR1-3), FGF receptors 1 and 2 (FGFR1-2) and ANG receptor (TIE2) was evaluated with the Western blot (WB) method. We also analysed the influence of chemerin on the phosphorylation of AMPK using WB. We found that in the studied endometrial samples, chemerin up-regulated the secretion of VEGF-A, VEGF-B and PlGF, and protein expression of VEGFR3. The adipokine caused a decrease in VEGF-C, VEGF-D and ANG-1 release. Chemerin effect on bFGF and ANG-2 secretion, and protein content of VEGFR1, VEGFR2, FGFR1, FGFR2 and TIE2 were dependent on the stage of pregnancy. Chemerin was found to down-regulate AMPK phosphorylation. The obtained in vitro results suggest that chemerin could be an important factor in the early pregnant uterus by its influence on angiogenic factors' secretion and signalling. The obtained results on the role of chemerin in the process of endometrial angiogenesis may, in the long term perspective, contribute to the elaboration of more effective methods of modifying reproductive processes and maintaining energy homeostasis in farm animals.