Enhanced AMPK Phosphorylation Research Articles

Acupuncture modulates the AMPK/PGC-1 signaling pathway to facilitate mitochondrial biogenesis and neural recovery in ischemic stroke rats

AimsThe main objective of this study was to investigate the role and mechanism of acupuncture on anti-nerve injury in the acute phase by regulating mitochondrial energy metabolism via monophosphate-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) axis in rat ischemic stroke.Main methodsMiddle cerebral artery occlusion (MCAO) was established by middle cerebral artery occlusion/reperfusion. One-week of acupuncture was performed during the acute phase of ischemic stroke. The neurological function and brain tissue integrity were evaluated. Mitochondrial function (intracellular ATP level and the activity of mitochondrial respiratory chain complex I) and the level of NADH oxidase (NOX) were detected by enzymatic chemistry. Next, the potential molecular mechanisms were explored by western blotting, fluorescence quantitative PCR and immunohistochemistry method.Key findings(1) Acupuncture treatment for MCAO/R rats showed a significant improvement in the infarcted tissue accompanied by functional recovery in Zea-Longa score and balance beam score outcomes, motor function performances. (2) Acupuncture increased the levels of ATP and mitochondrial respiratory chain complex I, decreased the NOX levels in cerebral ischemia established by suture-occluded method. (3) Acupuncture reduced the necrosis dissolution of neuronal cells and meningeal edema, while promoting angiogenesis. (4) Quantitative immunohistochemical staining results showed acupuncture can increase the expression of AMPK, p-AMPK and the mitochondrial transcription factor PGC-1α, NRF2, TFAM and uncoupling protein 2 (UCP2). Meanwhile, acupuncture treatment up-regulated the expression of the corresponding protein. (5) Subsequently, acupuncture enhanced AMPK phosphorylation as well as the expression of PGC-1α, NRF2, TFAM and UCP2, implicated in mitochondrial synthesis and cellular apoptosis. (6) Finally, injections of AMPK antagonists and activators confirmed AMPK as a therapeutic target for the anti-nerve damage effects of acupuncture.SignificanceAcupuncture intervention relieved ischemic stroke progression in MCAO rats by promoting energy metabolism and mitochondrial biogenesis in the brain and alleviating neuronal apoptosis, which was mediated by eliciting AMPK/PGC-1α axis, among them AMPK is a therapeutic target.

Angelica gigas extract inhibits acetylation of eNOS via IRE1α sulfonation/RIDD-SIRT1-mediated posttranslational modification in vascular dysfunction.

Angelica gigas NAKAI (AG) is a popular traditional medicinal herb widely used to treat dyslipidemia owing to its antioxidant activity. Vascular disease is intimately linked to obesity-induced metabolic syndrome, and AG extract (AGE) shows beneficial effects on obesity-associated vascular dysfunction. However, the effectiveness of AGE against obesity and its underlying mechanisms have not yet been extensively investigated. In this study, 40 high fat diet (HFD) rats were supplemented with 100-300 mg/kg/day of AGE to determine its efficacy in regulating vascular dysfunction. The vascular relaxation responses to acetylcholine were impaired in HFD rats, while the administration of AGE restored the diminished relaxation pattern. Endothelial dysfunction, including increased plaque area, accumulated reactive oxygen species, and decreased nitric oxide (NO) and endothelial nitric oxide synthase (eNOS) Ser1177 phosphorylation, were observed in HFD rats, whereas AGE reversed endothelial dysfunction and its associated biochemical signaling. Furthermore, AGE regulated endoplasmic reticulum (ER) stress and IRE1α sulfonation and its subsequent sirt1 RNA decay through controlling regulated IRE1α-dependent decay (RIDD) signaling, ultimately promoting NO bioavailability via the SIRT1-eNOS axis in aorta and endothelial cells. Independently, AGE enhanced AMPK phosphorylation, additionally stimulating SIRT1 and eNOS deacetylation and its associated NO bioavailability. Decursin, a prominent constituent of AGE, exhibited a similar effect in alleviating endothelial dysfunctions. These data suggest that AGE regulates dyslipidemia-associated vascular dysfunction by controlling ROS-associated ER stress responses, especially IRE1α-RIDD/sirt1 decay and the AMPK-SIRT1 axis.

Echinochrome A Prevents Diabetic Nephropathy by Inhibiting the PKC-Iota Pathway and Enhancing Renal Mitochondrial Function in db/db Mice.

Echinochrome A (EchA) is a natural bioproduct extracted from sea urchins, and is an active component of the clinical drug, Histochrome®. EchA has antioxidant, anti-inflammatory, and antimicrobial effects. However, its effects on diabetic nephropathy (DN) remain poorly understood. In the present study, seven-week-old diabetic and obese db/db mice were injected with Histochrome (0.3 mL/kg/day; EchA equivalent of 3 mg/kg/day) intraperitoneally for 12 weeks, while db/db control mice and wild-type (WT) mice received an equal amount of sterile 0.9% saline. EchA improved glucose tolerance and reduced blood urea nitrogen (BUN) and serum creatinine levels but did not affect body weight. In addition, EchA decreased renal malondialdehyde (MDA) and lipid hydroperoxide levels, and increased ATP production. Histologically, EchA treatment ameliorated renal fibrosis. Mechanistically, EchA suppressed oxidative stress and fibrosis by inhibiting protein kinase C-iota (PKCι)/p38 mitogen-activated protein kinase (MAPK), downregulating p53 and c-Jun phosphorylation, attenuating NADPH oxidase 4 (NOX4), and transforming growth factor-beta 1 (TGFβ1) signaling. Moreover, EchA enhanced AMPK phosphorylation and nuclear factor erythroid-2-related factor 2 (NRF2)/heme oxygenase 1 (HO-1) signaling, improving mitochondrial function and antioxidant activity. Collectively, these findings demonstrate that EchA prevents DN by inhibiting PKCι/p38 MAPK and upregulating the AMPKα/NRF2/HO-1 signaling pathways in db/db mice, and may provide a therapeutic option for DN.

Canagliflozin-loaded chitosan-hyaluronic acid microspheres modulate AMPK/NF-κB/NLRP3 axis: A new paradigm in the rectal therapy of ulcerative colitis

Ulcerative colitis is an idiopathic disease that is widely incident worldwide. Canagliflozin, antidiabetic agent, exhibited significant anti-inflammatory effects in a variety of animal models. Additionally, hyaluronic acid is considered one of the key players in the tissue regeneration process. It has been proven to modulate inflammation and cellular migration, which are the main phases of wound healing. The combination of hyaluronic acid with chitosan in microsphere fabrication was anticipated to reveal a synergistic muco-adhesiveness potential with additional advantage of the chitosan penetration enhancing effect. The current study aimed to explore the potential of canagliflozin-loaded chitosan-hyaluronic acid microspheres intrarectal administration to mitigate acetic acid-induced colitis in rats. Colon tissues were examined for macroscopic and microscopic pathological changes. ELISA and qRT-PCR techniques were applied for the detection of cytokines involved in the AMPK/NF-κB/NLRP3 axis. Intrarectal administration of this formula alleviated colitis severity, which was reflected by the reduced DAI, MES, colonic weight/length ratio and histopathological scoring values. Interestingly, canagliflozin-loaded chitosan-hyaluronic acid microspheres significantly enhanced AMPK phosphorylation and depressed NF-κB and NLRP3 expression leading to a subsequent reduction in caspase-1 cleavage and the inhibition of several inflammatory cytokines, including IL-1β, and IL-18. Overall, the current study revealed that the protective effects of the formula against acetic acid-induced colitis are primarily mediated via augmenting AMPK phosphorylation and its consequences of NF-κB inactivation. Since canagliflozin is not associated with hypoglycemic effects, clinical application of canagliflozin-loaded chitosan-hyaluronic acid microspheres represent a novel therapeutic option for the treatment of patients with ulcerative colitis.

Stachydrine alleviates lipid-induced skeletal muscle insulin resistance via AMPK/HO-1-mediated suppression of inflammation and endoplasmic reticulum stress.

Insulin resistance develops due to skeletal muscle inflammation and endoplasmic reticulum (ER) stress. Stachydrine (STA), extracted from Leonurus heterophyllus, has been shown to suppress proliferation and induce apoptosis in breast cancer cells and exert anti-inflammatory properties in the brain, heart, and liver. However, the roles of STA in insulin signaling in skeletal muscle remain unclear. Herein, we examined the impacts of STA on insulin signaling in skeletal muscle under hyperlipidemic conditions and its related molecular mechanisms. Various protein expression levels were determined by Western blotting. Levels of mouse serum cytokines were measured by ELISA. We found that STA-ameliorated inflammation and ER stress, leading to attenuation of insulin resistance in palmitate-treated C2C12 myocytes. STA dose-dependently enhanced AMPK phosphorylation and HO-1 expression. Administration of STA attenuated not only insulin resistance but also inflammation and ER stress in the skeletal muscle of high-fat diet (HFD)-fed mice. Additionally, STA-ameliorated glucose tolerance and insulin sensitivity, as well as serum TNFα and MCP-1, in mice fed a HFD. Small interfering (si) RNA-associated suppression of AMPK or HO-1 expression abolished the effects of STA in C2C12 myocytes. These results suggest that STA activates AMPK/HO-1 signaling, resulting in reduced inflammation and ER stress, thereby improving skeletal muscle insulin resistance. Using STA as a natural ingredient, this research successfully treated insulin resistance and type 2 diabetes.

Alginate Oligosaccharides Ameliorate DSS-Induced Colitis through Modulation of AMPK/NF-κB Pathway and Intestinal Microbiota.

Alginate oligosaccharides (AOS) are shown to have various biological activities of great value to medicine, food, and agriculture. However, little information is available about their beneficial effects and mechanisms on ulcerative colitis. In this study, AOS with a polymerization degree between 2 and 4 were found to possess anti-inflammatory effects in vitro and in vivo. AOS could decrease the levels of nitric oxide (NO), IL-1β, IL-6, and TNFα, and upregulate the levels of IL-10 in both RAW 264.7 and bone-marrow-derived macrophage (BMDM) cells under lipopolysaccharide (LPS) stimulation. Additionally, oral AOS administration could significantly prevent bodyweight loss, colonic shortening, and rectal bleeding in dextran sodium sulfate (DSS)-induced colitis mice. AOS pretreatment could also reduce disease activity index scores and histopathologic scores and downregulate proinflammatory cytokine levels. Importantly, AOS administration could reverse DSS-induced AMPK deactivation and NF-κB activation in colonic tissues, as evidenced by enhanced AMPK phosphorylation and p65 phosphorylation inhibition. AOS could also upregulate AMPK phosphorylation and inhibit NF-κB activation in vitro. Moreover, 16S rRNA gene sequencing of gut microbiota indicated that supplemental doses of AOS could affect overall gut microbiota structure to a varying extent and specifically change the abundance of some bacteria. Medium-dose AOS could be superior to low- or high-dose AOS in maintaining remission in DSS-induced colitis mice. In conclusion, AOS can play a protective role in colitis through modulation of gut microbiota and the AMPK/NF-kB pathway.

Angiotensin II Promotes White Adipose Tissue Browning and Lipolysis in Mice.

Emerging evidence has revealed that all components of the renin-angiotensin system (RAS) are present in adipose tissue. Angiotensin II (Ang II), the major bioactive component of the RAS, has been recognized as an adipokine involved in regulating energy homeostasis. However, the precise role of Ang II in white adipose tissue (WAT) remodeling remains to be elucidated. In this present study, C57BL/C male mice were continuously infused with different doses of Ang II (1.44 mg/kg/d or 2.5 mg/kg/d) or saline for 2 weeks and treated with or without the Ang II type 1 receptor blocker valsartan. H&E staining and immunohistochemistry were conducted to investigate the white-to-brown fat conversion. The level of serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) was measured. RNA sequencing was employed to explore the differentially expressed genes and their enriched pathways between control and Ang II groups. Our results showed that Ang II substantially resulted in loss of body weight and fat mass. Most importantly, Ang II treatment induced WAT browning in mice, which was partially attenuated by valsartan treatment. Furthermore, Ang II perturbed the serum lipid profiles. Ang II treatment elevated serum levels of TC, TG, LDL-C, and HDL-C in mice. Mechanistically, thermogenesis, cell respiration, and lipid metabolism-associated mRNAs showed significantly increased expression profiling in Ang II-treated WATs compared with control WATs. Moreover, we found that Ang II treatment enhanced AMPK phosphorylation in adipocytes. Therefore, Ang II promotes WAT browning and lipolysis via activating the AMPK signaling pathway.

Bak instead of Bax plays a key role in metformin-induced apoptosis s in HCT116 cells

Metformin (Met) exhibits anticancer ability in various cancer cell lines. This report aims to explore the exact molecular mechanism of Met-induced apoptosis in HCT116 cells, a human colorectal cancer cell line. Met-induced reactive oxygen species (ROS) increase and ROS-dependent cell death accompanied by plasma membrane blistering, mitochondrial swelling, loss of mitochondrial membrane potential, and release of cytochrome c. Western blotting analysis showed that Met upregulated Bak expression but downregulated Bax expression. Most importantly, silencing Bak instead of Bax inhibited Met-induced loss of mitochondrial membrane potential, indicating the key role of Bak in Met-induced apoptosis. Live-cell fluorescence resonance energy transfer (FRET) analysis showed that Met unlocked the binding of Mcl-1 to Bak, and enhanced the binding of Bim to Bak and subsequent Bak homo-oligomerization. Western blotting analysis showed that Met enhanced AMPK phosphorylation and Bim expression, and compound C, an inhibitor of AMPK, inhibited Met-induced Bim upregulation. Although Met increased the expression of Bcl-xL, overexpression of Bcl-xL did not prevent Met-induced apoptosis. In summary, our data demonstrate for the first time that Met promotes ROS-dependent apoptosis by regulating the Mcl-1-Bim-Bak axis.

Betulinic acid induces autophagy-dependent apoptosis via Bmi-1/ROS/AMPK-mTOR-ULK1 axis in human bladder cancer cells.

Betulinic acid (BA), a pentacyclic triterpenoid isolated from tree bark, exhibits antitumor effects against solid malignancies and triggers autophagy and/or apoptosis in human cancer cells. Nonetheless, the relationship between autophagy and apoptosis and the potential modulatory actions of BA on autophagy-dependent bladder cancer cell death remain unclear. The present study showed that BA exposure significantly suppressed viability, proliferation, and migration of EJ and T24 human bladder cancer cells. These effects reflected caspase 3-mediated apoptosis and could be attenuated or abolished by inhibiting ROS production with N-acetyl-L-cysteine, inhibiting autophagy with chloroquine, or silencing ATG7 with targeted siRNA. BA-induced autophagy was evidenced by epifluorescence imaging of lentivirus-induced expression of mCherry-GFP-LC3B and increased expression of two autophagy-related proteins, LC3B-II and TEM. Moreover, enhanced AMPK phosphorylation and decreased mTOR and ULK-1 phosphorylation suggested BA activates autophagy via the AMPK/mTOR/ULK1 pathway. Accordingly, exposure to dorsomorphin (Compound C), an AMPK inhibitor, and AICAR, an AMPK activator, respectively inhibited and stimulated BA-induced autophagy in EJ and T24 cells. The effects of Bmi-1 overexpression in vitro and decreased Bmi-1 expression in BA-treated T24 cell xenografts in nude mice suggested that downregulation of Bmi-1 is the underlying mechanism in BA-mediated, autophagy-dependent apoptosis.

3-hydroxymorphinan enhances mitochondrial biogenesis and adipocyte browning through AMPK-dependent pathway

3-hydroxymorphinan (3-HM), a metabolite of dextromethorphan, has previously been reported to have anti-inflammatory, anti-oxidative stress, and neuroprotective effects. However, its effect on energy metabolism in adipocytes remains unclear. Herein, we investigated 3-hydroxymorphinan (3-HM) effects on mitochondrial biogenesis, oxidative stress, and lipid accumulation in 3T3-L1 adipocytes. Further, we explored 3-HM-associated molecular mechanisms. Mouse adipocyte 3T3-L1 cells were treated with 3-HM, and various protein expression levels were determined by western blotting analysis. Mitochondria accumulation and lipid accumulation were measured by staining methods. Cell toxicity was assessed by cell viability assay. We found that treatment of 3T3-L1 adipocytes with 3-HM increased expression of brown adipocyte markers, such as uncoupling protein-1 (UCP-1) and peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α). 3-HM promotes mitochondrial biogenesis and its-mediated gene expression. Additionally, 3-HM treatment suppressed mitochondrial ROS generation and superoxide along with improved mitochondrial complex I activity. We found that treatment of 3-HM enhanced AMPK phosphorylation. siRNA-mediated suppression of AMPK reversed all these changes in 3T3-L1 adipocytes. In sum, 3-HM promotes mitochondrial biogenesis and browning and attenuates oxidative stress and lipid accumulation in 3T3-L1 adipocytes via AMPK signaling. Thus, 3-HM-mediated AMPK activation can be considered a therapeutic approach for treating obesity and related diseases.

Interference With the AMPKα/mTOR/NLRP3 Signaling and the IL-23/IL-17 Axis Effectively Protects Against the Dextran Sulfate Sodium Intoxication in Rats: A New Paradigm in Empagliflozin and Metformin Reprofiling for the Management of Ulcerative Colitis.

Empagliflozin and metformin are widely used for the treatment of type 2 diabetes. These drugs showed marked anti-inflammatory effects in different animal models via enhancing AMPK activity. Yet, the protective anti-inflammatory effects of their combination against ulcerative colitis have not been previously investigated. The current study aimed to explore the potential of empagliflozin/metformin combination to mitigate the DSS-induced rat colitis model. The modulating effects of empagliflozin and metformin on the AMPK/mTOR/NLRP3 axis and T cell polarization were delineated. In this study, distal colons were examined for macroscopic and microscopic pathological alterations. ELISA, qRT-PCR, and immunohistochemistry techniques were applied to detect proteins and cytokines involved in AMPK/mTOR/NLRP3 axis and T Cell polarization. Oral administration of empagliflozin (10 mg/kg/day) and metformin (200 mg/kg/day) combination alleviated colitis as revealed by the reduced disease activity index, macroscopic damage index, colon weight/length ratio, and histopathologic scoring values. Interestingly, empagliflozin/metformin combination significantly enhanced AMPK phosphorylation and depressed mTOR and NLRP3 expression leading to a subsequent reduction in caspase-1 cleavage and inhibition of several inflammatory cytokines, including IL-1β, and IL-18. Reduced mTOR expression and reduced IL-6 levels led to a reduction in Th17 cell polarization and maintenance. Together, the current study reveals that the protective effects of empagliflozin and metformin against DSS-induced colitis are fundamentally mediated via enhancing AMPK phosphorylation. Since adult humans with diabetes mellitus are at greater risk for developing inflammatory bowel diseases, clinical application of empagliflozin/metformin combination represents a novel therapeutic approach for treating diabetic patients with ulcerative colitis.

Meteorin-like protein (METRNL)/IL-41 improves LPS-induced inflammatory responses via AMPK or PPARδ–mediated signaling pathways

PurposeMeteorin-like protein (METRNL) (also known as IL-41), recently identified as a myokine, is released in response to muscle contraction. It improves the skeletal muscle insulin sensitivity through exerting a beneficial anti-inflammatory effect. However, no independent studies have been published to verify the effects of METRNL on human umbilical vein endothelial cells (HUVECs) and THP-1 human monocytes. Materials and methodsThe levels of NFκB and IκB phosphorylation as well as the expression of adhesion molecules were assessed by Western blotting analysis. Cell adhesion assay demonstrated the interactions between HUVEC and THP-1 ​cells. We used enzyme-linked immunosorbent assay (ELISA) to measure the levels of TNFα and MCP-1 in culture medium. ResultsTreatment with METRNL suppressed the secretion of TNFα and MCP-1 as well as NFκB and IκB phosphorylation and inflammatory markers in lipopolysaccharide (LPS)-treated HUVECs and THP-1 ​cells. Furthermore, treatment with METRNL ameliorated LPS-induced attachment of THP-1 monocytes to HUVECs via inhibition of adhesion molecule expression and apoptosis. Treatment of HUVEC and THP-1 ​cells with METRNL enhanced AMPK phosphorylation and PPARδ expression in a dose-dependent manner. Small interference (si) RNA-mediated suppression of AMPK or PPARδ restored all these changes. ConclusionsIt has therefore been shown that METRNL ameliorates inflammatory responses through AMPK and PPARδ-dependent pathways in LPS-treated HUVEC. In sum, the current study may suggest the suppressive potential of METRNL against endothelial inflammation.

Extracellular ATP modulates podocyte function through P2Y purinergic receptors and pleiotropic effects on AMPK and cAMP/PKA signaling pathways

Podocytes and their foot processes interlinked by slit diaphragms, constitute a continuous outermost layer of the glomerular capillary and seem to be crucial for maintaining the integrity of the glomerular filtration barrier. Purinergic signaling is involved in a wide range of physiological processes in the renal system, including regulating glomerular filtration. We evaluated the role of nucleotide receptors in cultured rat podocytes using non-selective P2 receptor agonists and agonists specific for the P2Y1, P2Y2, and P2Y4 receptors. The results showed that extracellular ATP evokes cAMP-dependent pathways through P2 receptors and influences remodeling of the podocyte cytoskeleton and podocyte permeability to albumin via coupling with RhoA signaling. Our findings highlight the relevance of the P2Y4 receptor in protein kinase A–mediated signal transduction to the actin cytoskeleton. We observed increased cAMP concentration and decreased RhoA activity after treatment with a P2Y4 agonist. Moreover, protein kinase A inhibitors reversed P2Y4-induced changes in RhoA activity and intracellular F-actin staining. P2Y4 stimulation resulted in enhanced AMPK phosphorylation and reduced reactive oxygen species generation. Our findings identify P2Y–PKA–RhoA signaling as the regulatory mechanism of the podocyte contractile apparatus and glomerular filtration. We describe a protection mechanism for the glomerular barrier linked to reduced oxidative stress and reestablished energy balance.

Neuroprotective effect of cajaninstilbene acid against cerebral ischemia and reperfusion damages by activating AMPK/Nrf2 pathway

IntroductionIschemic stroke is one of the leading causes of death worldwide. Recently, neuroprotection is regarded as an important preventative and therapeutic strategy for ischemic stroke. Cajaninstilbene acid (CSA), a unique stilbenoid with a styryl group, is a potential neuroprotective agent. ObjectivesHence, this study aimed to evaluate the neuroprotective effect and molecular mechanism of CSA against cerebral ischemia/reperfusion (I/R) damages. MethodsCerebral ischemia was modeled by oxygen and glucose deprivation (OGD) in SH-SY5Y cells or transient intraluminal suture middle cerebral artery occlusion (MCAO) in rats, and tert-butyl hydroperoxide (t-BHP) was used to induce oxidative stress in SH-SY5Y cells. CSA (2.5, 5 mg/kg) was intraperitoneally given upon reperfusion after 2 h of MCAO. The signaling pathways were analyzed by Western blotting and inhibitor blocking. ResultsCSA possessed significant neuroprotective activity, as evidenced by the reduced cell death in OGD/R or t-BHP injured SH-SY5Y cells, and decreased infarct volume and neurological deficits in MCAO/R rats. Further studies indicated that the protective effect was achieved via the antioxidant activity of CSA, which decreased the oxidative stress and its related mitochondrial dysfunction in SH-SY5Y cells. Notably, Nrf2 was activated in SH-SY5Y cells and MCAO/R rats by CSA, and the inhibition of Nrf2 by brusatol weakened CSA-mediated neuroprotection. Furthermore, after applying a series of kinase inhibitors, CSA-induced Nrf2 activation was markedly inhibited by BML-275 (an AMPK inhibitor), implying that AMPK was the dominant kinase to regulate the Nrf2 pathway for CSA’s neuroprotective effects with enhanced AMPK phosphorylation observed both in vivo and in vitro. ConclusionCSA exerted neuroprotection via activating the AMPK/Nrf2 pathway to reduce I/R-induced cellular oxidative stress and mitochondrial disfunction. CSA could be a potential neuroprotective drug candidate for the treatment of ischemic stroke.

Effect of oleuropein on oxidative stress, inflammation and apoptosis induced by ischemia-reperfusion injury in rat kidney

AimsThis study aimed to evaluate the effect of oleuropein (OLE), the main phenolic compound present in olive leaves, on kidney ischemia-reperfusion injury (IRI) and to explore the underlying protective mechanism. Main methodsRat kidneys were subjected to 60 min of bilateral warm ischemia followed by 120 min of reperfusion. OLE was administered orally 48 h, 24 h and 30 min prior to ischemia at doses of 10, 50 and 100 mg/kg body weight. The creatinine, urea, uric acid concentrations and lactate dehydrogenase (LDH) activity in plasma were evaluated. Oxidative stress and inflammation parameters were also assessed. Renal expression of AMP-activated protein kinase (p-AMPK), endothelial nitric oxide synthase (eNOS), mitogen-activated protein kinases (MAPK), inflammatory proteins and apoptotic proteins were evaluated using Western blot. Key findingsOur results showed that OLE at 50 mg/kg reduced kidney IRI as revealed by a significant decrease of plasmatic creatinine, urea, uric acid concentrations and LDH activity. In parallel, OLE up-regulated antioxidant capacities. Moreover, OLE diminished the level of CRP and the expression of cyclooxygenase 2 (COX-2). Finally, OLE enhanced AMPK phosphorylation as well as eNOS expression whereas MAPK, and cleaved caspase-3 implicated in cellular apoptosis were attenuated in the ischemic kidneys. SignificanceIn conclusion, this study shows that OLE could be used as therapeutic agent to reduce IRI through its anti-oxidative, anti-inflammatory and anti-apoptotic properties.

Humanin attenuates palmitate-induced hepatic lipid accumulation and insulin resistance via AMPK-mediated suppression of the mTOR pathway

The pathogenesis of non-alcoholic fatty liver disease (NAFLD) remains unclear. Humanin (HN), a cytoprotective polypeptide, reportedly exhibits neuroprotective effects via suppression of inflammation and improvement of insulin resistance in neurons. This study aim was to investigate effects of HN on lipid accumulation in the hepatocytes and insulin signaling, and explore the underlying mechanisms. Protein expression levels were analyzed by Western blotting. Hepatic lipid accumulation was confirmed by Oil red-O staining. We found that HN-treatment ameliorated palmitate-induced lipid accumulation, expression of lipogenesis-associated genes (processed SREBP1, FAS, and SCD1), cell death, and caspase 3 activity in hepatocytes in a dose-dependent manner. Additionally, HN attenuated palmitate-induced impairment of insulin signaling. HN enhanced AMPK phosphorylation, whereas it suppressed palmitate-induced phosphorylation of mTOR. AMPK knockdown by siRNA neutralized the effects of HN on palmitic acid-treated hepatocytes. Collectively, HN prevents palmitate-induced hepatic lipid accumulation, apoptosis, and insulin resistance via AMPK-mediated suppression of the mTOR/SREBP1 pathway, suggesting that it may serve as a potential therapeutic agent in NAFLD treatment.

Taurine-mediated browning of white adipose tissue is involved in its anti-obesity effect in mice

Taurine, a nonprotein amino acid, is widely distributed in almost all animal tissues. Ingestion of taurine helps to improve obesity and its related metabolic disorders. However, the molecular mechanism underlying the protective role of taurine against obesity is not completely understood. In this study, it was found that intraperitoneal treatment of mice with taurine alleviated high-fat diet (HFD)-induced obesity, improved insulin sensitivity, and increased energy expenditure and adaptive thermogenesis of the mice. Meanwhile, administration of the mice with taurine markedly induced the browning of inguinal white adipose tissue (iWAT) with significantly elevated expression of PGC1α, UCP1, and other thermogenic genes in iWAT. In vitro studies indicated that taurine also induced the development of brown-like adipocytes in C3H10T1/2 white adipocytes. Knockdown of PGC1α blunted the role of taurine in promoting the brown-like adipocyte phenotypes in C3H10T1/2 cells. Moreover, taurine treatment enhanced AMPK phosphorylation in vitro and in vivo, and knockdown of AMPKα1 prevented taurine-mediated induction of PGC1α in C3H10T1/2 cells. Consistently, specific knockdown of PGC1α in iWAT of the HFD-fed mice inhibited taurine-induced browning of iWAT, with the role of taurine in the enhancement of adaptive thermogenesis, the prevention of obesity, and the improvement of insulin sensitivity being partially impaired. These results reveal a functional role of taurine in facilitating the browning of white adipose tissue, which depends on the induction of PGC1α. Our studies also suggest a potential mechanism for the protective role of taurine against obesity, which involves taurine-mediated browning of white adipose tissue.

Oxidative stress-mediated TXNIP loss causes RPE dysfunction

The disruption of the retinal pigment epithelium (RPE), for example, through oxidative damage, is a common factor underlying age-related macular degeneration (AMD). Aberrant autophagy also contributes to AMD pathology, as autophagy maintains RPE homeostasis to ensure blood–retinal barrier (BRB) integrity and protect photoreceptors. Thioredoxin-interacting protein (TXNIP) promotes cellular oxidative stress by inhibiting thioredoxin reducing capacity and is in turn inversely regulated by reactive oxygen species levels; however, its role in oxidative stress-induced RPE cell dysfunction and the mechanistic link between TXNIP and autophagy are largely unknown. Here, we observed that TXNIP expression was rapidly downregulated in RPE cells under oxidative stress and that RPE cell proliferation was decreased. TXNIP knockdown demonstrated that the suppression of proliferation resulted from TXNIP depletion-induced autophagic flux, causing increased p53 activation via nuclear localization, which in turn enhanced AMPK phosphorylation and activation. Moreover, TXNIP downregulation further negatively impacted BRB integrity by disrupting RPE cell tight junctions and enhancing cell motility by phosphorylating, and thereby activating, Src kinase. Finally, we also revealed that TXNIP knockdown upregulated HIF-1α, leading to the enhanced secretion of VEGF from RPE cells and the stimulation of angiogenesis in cocultured human retinal microvascular endothelial cells. This suggests that the exposure of RPE cells to sustained oxidative stress may promote choroidal neovascularization, another AMD pathology. Together, these findings reveal three distinct mechanisms by which TXNIP downregulation disrupts RPE cell function and thereby exacerbates AMD pathogenesis. Accordingly, reinforcing or restoring BRB integrity by targeting TXNIP may serve as an effective therapeutic strategy for preventing or attenuating photoreceptor damage in AMD.

Adrenomedullin alleviates the pyroptosis of Leydig cells by promoting autophagy via the ROS\u2013AMPK\u2013mTOR axis

Adrenomedullin (ADM) exerts anti-oxidant, anti-inflammatory and anti-apoptotic effects in Leydig cells. However, the role and mechanism of ADM in the pyroptosis of Leydig cells are poorly understood. This study first showed the protective effects of ADM on the pyroptosis and biological functions of Leydig cells exposed to lipopolysaccharide (LPS) by promoting autophagy. Primary rat Leydig cells were treated with various concentrations of LPS and ADM, together with or without N-acetyl-L-cysteine (NAC) or 3-methyladenine (3-MA). Cell proliferation was detected through CCK-8 and BrdU incorporation assays, and ROS level was measured with the DCFDA assay. Real-time PCR, western blot, immunofluorescence, transmission electron microscopy, TUNEL and flow cytometry were performed to examine ADM’s effect on the pyroptosis, autophagy and steroidogenic enzymes of Leydig cells and AMPK/mTOR signalling. Like NAC, ADM dose-dependently reduced LPS-induced cytotoxicity and ROS overproduction. ADM also dose-dependently ameliorated LPS-induced pyroptosis by reversing the increased expression of NLRP3, ASC, caspase-1, IL-1β, IL-18, GSDMD, caspase-3, caspase-7, TUNEL-positive and PI and active caspase-1 double-stained positive rate, DNA fragmentation and LDH concentration, which could be rescued via co-incubation with 3-MA. ADM dose-dependently increased autophagy in LPS-induced Leydig cells, as confirmed by the increased expression of LC3-I/II, Beclin-1 and ATG-5; decreased expression of p62 and autophagosomes formation; and increased LC3-II/LC3-I ratio. However, co-treatment with 3-MA evidently decreased autophagy. Furthermore, ADM dose-dependently rescued the expression of steroidogenic enzymes, including StAR, P450scc, 3β-HSD and CYP17, and testosterone production in LPS-induced Leydig cells. Like rapamycin, ADM dose-dependently enhanced AMPK phosphorylation but reduced mTOR phosphorylation in LPS-induced Leydig cells, which could be rescued via co-incubation with 3-MA. In addition, pyroptosis was further decreased, and autophagy was further promoted in LPS-induced Leydig cells upon co-treatment with ADM and rapamycin. ADM may protect the steroidogenic functions of Leydig cells against pyroptosis by activating autophagy via the ROS–AMPK–mTOR axis.

166-LB: Canagliflozin but Not Empagliflozin Activates AMPK and Suppresses Colon Cancer Cell Growth

Background: Recent increase in the relative proportion of cancer death in diabetic patients has become a major issue. A hypoglycemic agent, sodium-glucose cotransporter (SGLT) 2 inhibitor has attracted attention because of its potency in reducing cardiovascular risks. Several lines of evidence has emerged suggesting a potential anti-oncotic effect. Here we address potential effects of canagliflozin (Cana) on human colon cancer cell growth and underlying mechanisms and compared with empagliflozin (Empa). Material and Methods: We examined the effects of Cana and Empa on 1) cell proliferation using colon cancer cell lines, SW480 and HCT116 by MTT assay, 2) DNA synthesis by flow cytometry-based analysis of bromodeoxyuridine (BrdU) uptake. We further examined 3) the expression of SGLT1 and 2 in these cells and 4) investigated molecules of signal transduction pathways with established roles in cancer cell growth such as AMPK, 4EBP1, mTOR by western blotting. Finally, we analyzed 5) time-dependent changes of metabolomic signatures after the treatment with Cana. Results: While SGLT1 and 2 mRNA and protein expression was detected in both cell lines, Cana but not Empa significantly inhibited cell proliferation and in a dose-dependent manner. Cana suppressed BrdU positive cells and enhanced AMPK phosphorylation with a consequent suppression of 4EBP1 and mTOR activity. Metabolomic profiling revealed that, as a function of time, intermediate metabolites of glycolysis especially phosphoenolpyruvate was decreased with an acute rise in ADP and gradual decrease in ATP and GSSG. Conclusion: We demonstrate that Cana deprives human colon cancer cells of energy and anti-oxidative capacity possibly through an inhibition of SGLT1-mediated glucose influx, and thereby activates AMPK and inhibits cancer cell growth. Our data herein suggest a potential beneficial effect of Cana in suppressing colon cancer progression especially in patients with diabetes. Disclosure T. Yagi: None. T. Tanaka: Research Support; Self; Japan Society for the Promotion of Science. Speaker's Bureau; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Kissei Pharmaceutical Co., Ltd., Kowa Pharmaceutical Europe Co. Ltd., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Taisho Toyama Pharmaceutical Co. Ltd., Takeda Pharmaceutical Company Limited. E. Kubota: None. H. Koyama: None. T. Guo: None. K. Ogawa: None. D. Aotani: None. H. Kataoka: None.