Activating AMP-activated Protein Kinase Signaling Research Articles

Mammalian target of differentiation-inducing factor-1 is mitochondrial malate dehydrogenase for activation of AMP-activated protein kinase and induction of mitochondrial fission

AimsDifferentiation-inducing factor-1 (DIF-1) is a polyketide produced by Dictyostelium discoideum that inhibits growth and migration, while promoting the differentiation of Dictyostelium stalk cells through unknown mechanisms. DIF-1 localizes in stalk mitochondria. In addition to its effect on Dictyostelium, DIF-1 also inhibits growth and migration, and induces mitochondrial fission followed by mitophagy in mammalian cells, at least in part by activating AMP-activated protein kinase (AMPK). In a previous study, we found that DIF-1 binds to mitochondrial malate dehydrogenase (MDH2) and inhibits its activity in HeLa cells. In the present study, we investigated whether MDH2 serves as a pharmacological target of DIF-1 in mammalian cells. Main methodsTo examine the enzymatic activity of MDH, mitochondrial morphology, and molecular mechanisms of DIF-1 action, we conducted an MDH reverse reaction assay, immunofluorescence staining, western blotting, and RNA interference using mammalian cells such as human umbilical vein endothelial cells, human cervical cancer cells, mouse endothelial cells, and mouse breast cancer cells. Key findingsDIF-1 inhibited mitochondrial but not cytoplasmic MDH activity. Similar to DIF-1, LW6, an authentic MDH2 inhibitor, induced phosphorylation of AMPK, resulting in the phosphorylation of acetyl-CoA carboxylase (ACC) and the dephosphorylation of p70 S6 kinase with approximately the same potency. DIF-1 and LW6 induced mitochondrial fission. Furthermore, MDH2 knockdown using siRNA reproduced the DIF-1 action on the AMPK signaling and mitochondrial morphology. Conversely, an AMPK inhibitor prevented DIF-1-induced mitochondrial fission. SignificanceWe propose that MDH2 is a mammalian target of DIF-1 for the activation of AMPK and induction of mitochondrial fission.

Melatonin Improves Glucose Homeostasis and Insulin Sensitivity by Mitigating Inflammation and Activating AMPK Signaling in a Mouse Model of Sleep Fragmentation.

Sleep fragmentation (SF) can increase inflammation and production of reactive oxygen species (ROS), leading to metabolic dysfunction. SF is associated with inflammation of adipose tissue and insulin resistance. Several studies have suggested that melatonin may have beneficial metabolic effects due to activating AMP-activated protein kinase (AMPK). However, it is unclear whether melatonin affects the AMPK signaling pathway in SF-induced metabolic dysfunction. Therefore, we hypothesize that SF induces metabolic impairment and inflammation in white adipose tissue (WAT), as well as altered intracellular homeostasis. We further hypothesize that these conditions could be improved by melatonin treatment. We conducted an experiment using adult male C57BL/6 mice, which were divided into three groups: control, SF, and SF with melatonin treatment (SF+Mel). The SF mice were housed in SF chambers, while the SF+Mel mice received daily oral melatonin. After 12 weeks, glucose tolerance tests, insulin tolerance tests, adipose tissue inflammation tests, and AMPK assessments were performed. The SF mice showed increased weight gain, impaired glucose regulation, inflammation, and decreased AMPK in WAT compared to the controls. Melatonin significantly improved these outcomes by mitigating SF-induced metabolic dysfunction, inflammation, and AMPK downregulation in adipose tissue. The therapeutic efficacy of melatonin against cardiometabolic impairments in SF may be due to its ability to restore adipose tissue homeostatic pathways.

LanGui tea, an herbal medicine formula, protects against binge alcohol-induced acute liver injury by activating AMPK-NLRP3 signaling

BackgroundLanGui tea, a traditional Chinese medicine formulation comprising of Gynostemma pentaphyllum (Thunb.) Makino, Cinnamomum cassia (L.) J. Presl, and Ampelopsis grossedentata (Hand-Mazz) W.T. Wang, has yet to have its potential contributions to alcoholic liver disease (ALD) fully elucidated. Consequently, the objective of this research is to investigate the protective properties of LanGui tea against binge alcohol-induced ALD and the mechanisms underlying its effects.MethodsAn experimental model of acute alcohol-induced liver disease was performed to assess the protective effects of extract of LanGui tea (ELG) at both 50 and 100 mg.kg−1 dosages on male C57BL/6 mice. Various parameters, including hepatic histological changes, inflammation, lipids content, as well as liver enzymes and interleukin 1β (IL-1β) in the serum were measured. The pharmacological mechanisms of ELG, specifically its effects on adenosine monophosphate-(AMP)-activated protein kinase (AMPK) and NLR family pyrin domain containing 3 (NLRP3) signaling, were investigated through Western blotting, qRT–PCR, ELISA, immunohistochemistry, immunofluorescence analyses, and by blocking the AMPK activity.ResultsELG demonstrated a mitigating effect on fatty liver, inflammation, and hepatic dysfunction within the mouse model. This effect was achieved by activating AMPK signaling and inhibitingNLRP3 signaling in the liver, causing a reduction in IL-1β generation. In vitro studies further confirmed that ELG inhibited cell damage and IL-1β production in ethanol-induced hepatocytes by enhancing AMPK-NLRP3 signaling. Conversely, the pharmacological inhibition of AMPK activity nearly abrogated such alteration.ConclusionsThus, LanGui tea emerges as a promising herbal therapy for ALD management involving AMPK-NLRP3 signaling.

Carnosic Acid against Lung Cancer: Induction of Autophagy and Activation of Sestrin-2/LKB1/AMPK Signalling.

Non-small cell lung cancer (NSCLC) represents 80% of all lung cancer cases and is characterized by low survival rates due to chemotherapy and radiation resistance. Novel treatment strategies for NSCLC are urgently needed. Liver kinase B1 (LKB1), a tumor suppressor prevalently mutated in NSCLC, activates AMP-activated protein kinase (AMPK) which in turn inhibits mammalian target of rapamycin complex 1 (mTORC1) and activates unc-51 like autophagy activating kinase 1 (ULK1) to promote autophagy. Sestrin-2 is a stress-induced protein that enhances LKB1-dependent activation of AMPK, functioning as a tumor suppressor in NSCLC. In previous studies, rosemary (Rosmarinus officinalis) extract (RE) activated the AMPK pathway while inhibiting mTORC1 to suppress proliferation, survival, and migration, leading to the apoptosis of NSCLC cells. In the present study, we investigated the anticancer potential of carnosic acid (CA), a bioactive polyphenolic diterpene compound found in RE. The treatment of H1299 and H460 NSCLC cells with CA resulted in concentration and time-dependent inhibition of cell proliferation assessed with crystal violet staining and 3H-thymidine incorporation, and concentration-dependent inhibition of survival, assessed using a colony formation assay. Additionally, CA induced apoptosis of H1299 cells as indicated by decreased B-cell lymphoma 2 (Bcl-2) levels, increased cleaved caspase-3, -7, poly (ADP-ribose) polymerase (PARP), Bcl-2-associated X protein (BAX) levels, and increased nuclear condensation. These antiproliferative and proapoptotic effects coincided with the upregulation of sestrin-2 and the phosphorylation/activation of LKB1 and AMPK. Downstream of AMPK signaling, CA increased levels of autophagy marker light chain 3 (LC3), an established marker of autophagy; inhibiting autophagy with 3-methyladenine (3MA) blocked the antiproliferative effect of CA. Overall, these data indicate that CA can inhibit NSCLC cell viability and that the underlying mechanism of action of CA involves the induction of autophagy through a Sestrin-2/LKB1/AMPK signaling cascade. Future experiments will use siRNA and small molecule inhibitors to better elucidate the role of these signaling molecules in the mechanism of action of CA as well as tumor xenograft models to assess the anticancer properties of CA in vivo.

Exerkine β-aminoisobutyric acid protects against atrial structural remodeling and atrial fibrillation in obesity via activating AMPK signaling and improving insulin sensitivity

Moderate exercise decreases the risk for atrial fibrillation (AF), an effect which is probably mediated via exercise-stimulated release of exerkines. β-Aminoisobutyric acid (BAIBA), a novel exerkine, has been reported to provide protective benefits against many cardiovascular diseases, yet its role in AF remains elusive. Herein, using a mouse model of obesity-related AF through high-fat diet (HFD) feeding, we found that 12-week drinking administration of BAIBA (170 mg/kg/day) decreased AF susceptibility in obese mice. Atrial remodeling assessment showed that BAIBA attenuated obesity-induced atrial hypertrophy and interstitial fibrosis, thereby ablating the substrate for AF. Of note, to our knowledge, this is the first report of the direct association of BAIBA and hypertrophy. BAIBA has been reported to be a key regulator of glucose and lipid metabolism, and we found that BAIBA alleviated insulin resistance in obese mice. Transcriptional analysis of metabolism-related genes showed that BAIBA increased the transcription of fatty acids metabolism-related genes in the atria of lean mice but not in that of obese mice. Mechanistic investigation showed that BAIBA stimulated AMP-activated protein kinase (AMPK) signaling in the atria of obese mice and palmitic acid (PA)-treated neonatal rat cardiomyocytes (NRCM), whereas inhibition of AMPK via Compound C attenuated BAIBA-conferred cardioprotection against hypertrophy and insulin resistance in PA-treated NRCM. Collectively, BAIBA attenuates AF susceptibility in obese mice via activated AMPK signaling and resultant improvement of insulin sensitivity, thereby providing perspectives on the potential therapeutic role of BAIBA in AF treatment.

Promotion of ROS-mediated apoptosis, G2/M arrest, and autophagy by naringenin in non-small cell lung cancer.

Background: As lung cancer is the leading cause of cancer death worldwide, the development of new medicines is a crucial endeavor. Naringenin, a flavanone derivative, possesses anti-cancer and anti-inflammatory properties and has been reported to have cytotoxic effects on various cancer cells. The current study investigated the underlying molecular mechanism by which naringenin induces cell death in lung cancer. Methods: The expression of apoptosis, cell cycle arrest, and autophagy markers in H1299 and A459 lung cancer cells was evaluated using a terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL), Western blot, Annexin V/PI stain, PI stain, acridine orange staining, and transmission electron microscopy (TEM). Using fluorescence microscopy, DALGreen was used to observe the degradation of p62, a GFP-LC3 plasmid was used to evaluate puncta formation, and a pcDNA3-GFP-LC3-RFP-LC3ΔG plasmid was used to evaluate autophagy flux. Furthermore, the anti-cancer effect of naringenin was evaluated in a subcutaneous H1299 cell xenograft model. Results: Naringenin treatment of lung cancer cells (H1299 and A459) reduced cell viability and induced cell cycle arrest. Pretreatment of cells with ROS scavengers (N-acetylcysteine or catalase) suppressed the naringenin-induced cleavage of apoptotic protein and restored cyclin-dependent kinase activity. Naringenin also triggered autophagy by mediating ROS generation, thereby activating AMP-activated protein kinase (AMPK) signaling. ROS inhibition not only inhibited naringenin-induced autophagic puncta formation but also decreased the ratio of microtubule-associated proteins 1A/1B light chain 3 II (LC3II)/LC3I and activity of the AMPK signaling pathway. Furthermore, naringenin suppressed tumor growth and promoted apoptosis in the xenograft mouse model. Conclusion: This study demonstrated the potent anti-cancer effects of naringenin on lung cancer cells, thereby providing valuable insights for developing small-molecule drugs that can induce cell cycle arrest, apoptosis, and autophagic cell death.

Follistatin-like 1 protects endothelial function in the spontaneously hypertensive rat by inhibition of endoplasmic reticulum stress through AMPK-dependent mechanism

ABSTRACT Objective Endothelial dysfunction is a critical initiating factor in the development of hypertension and related complications. Follistatin-like 1 (FSTL1) can promote endothelial cell function and stimulates revascularization in response to ischemic insult. However, it is unclear whether FSTL1 has an effect on ameliorating endothelial dysfunction in spontaneously hypertensive rats (SHRs). Methods Wistar Kyoto (WKY) and SHRs were treated with a tail vein injection of vehicle (1 mL/day) or recombinant FSTL1 (100 μg/kg body weight/day) for 4 weeks. Blood pressure was measured by tail-cuff plethysmograph, and vascular reactivity in mesenteric arteries was measured using wire myography. Results We found that treatment with FSTL1 reversed impaired endothelium-dependent relaxation (EDR) in mesenteric arteries and lowered blood pressure of SHRs. Decreased AMP-activated protein kinase (AMPK) phosphorylation, elevated endoplasmic reticulum (ER) stress markers, increased reactive oxygen species (ROS), and reduction of nitric oxide (NO) production in mesenteric arteries of SHRs were also reversed by FSTL1 treatment. Ex vivo treatment with FSTL1 improved the impaired EDR in mesenteric arteries from SHRs and reversed tunicamycin (ER stress inducer)-induced ER stress and the impairment of EDR in mesenteric arteries from WKY rats. The effects of FSTL1 were abolished by cotreatment of compound C (AMPK inhibitor). Conclusions These results suggest that FSTL1 prevents endothelial dysfunction in mesenteric arteries of SHRs through inhibiting ER stress and ROS and increasing NO production via activation of AMPK signaling.

CD19-CAR T Cytotoxicity Is Improved By AMPKγ2 Overexpression, Which Is Further Enhanced By Metformin Treatment

Background Acute lymphoblastic leukemia (ALL) is the most common leukemia found in children. Chimeric Antigen Receptor (CAR) T cells induce high response rates against relapsed/refractory B-cell ALL, but 40% of CAR-T recipients suffer disease relapse, many due to poor in vivo survival of the CAR-T cells. Metabolic exhaustion has been proposed as a major barrier to optimal CAR-T cell performance. Therefore, modulating T cell metabolism may represent a promising method to improve CAR T-cell therapy. AMP-activated protein kinase (AMPK) is a heterotrimeric signaling complex which serves as a cellular energy sensor, promoting mitochondrial health and oxidative metabolism under conditions of energetic stress. We have demonstrated overexpressing the regulatory subunit AMPKγ2 increases AMPK signaling in human T cells, and hypothesized that this overexpression would enhance CAR-T metabolic fitness and improve anti-leukemia activity. Results We used lentiviral transduction to introduce a CD19-reactive, CD28 CAR into human T cells, followed by transduction of either AMPKγ2 or an empty vector (EV) control. Dual transduced cells were isolated by flow sorting and cultured with human IL2. The metabolism and cytotoxicity of these CAR-T cells were first assessed in vitro. AMPK-CART cells showed higher oxidative metabolism, with a 29% increase in basal oxygen consumption rates (OCR) and a 45% increase in maximal OCR (p<0.001) following overnight stimulation by NALM6 cells. In addition, co-culture with Zs-Green+ NALM6 cells revealed greater cytotoxicity by AMPK-CART cells (p<0.001) (Fig. 1A), accompanied by a 35% increase in CD25 expression (p<0.05). We then pivoted to studying AMPK-CART cells in vivo utilizing a murine xenograft leukemia model. Interestingly, despite their in vitro advantages, AMPK-CART cells demonstrated equivalent anti-tumor capacity in vivo. We reasoned that AMPKγ2 overexpression may simply protect AMPK signaling which is already occurring. To test this idea, we coupled AMPKγ2 overexpression with transient metformin treatment, a process known to activate AMPK signaling through inhibition of mitochondrial complex I. Metformin treatment was followed by the functional analysis of CAR-T cells in a chronic stimulation protocol where we repetitively stimulate CAR-T cells with NALM6 targets to induce exhaustion. After 8 days of chronic stimulation, metformin treated AMPK-CART cells (7mM for 4 hours) demonstrated the highest cytotoxicity against NALM6 cells compared to either metformin treated EV-CART cells or AMPK-CART cells without metformin treatment (Fig. 1B). This cytotoxic advantage was accompanied by an 18% increase in CD25 expression (2052.67±104.74 vs. 1742.67±14.64, p<0.05) and a 56% elevation in the percentage of central memory cells (9.15±1.32 vs. 5.88±0.25, p<0.05) in metformin treated AMPK-CART cells. These changes were not seen in EV-CART cells with metformin treatment. Conclusions Here, we report that AMPKγ2 overexpression in CD19-CAR T cells enhances oxidative metabolism and improves in vitro cytotoxicity but does not significantly increase in vivo anti-leukemia function. Metformin pre-treatment elevates CAR-T cytotoxicity following chronic antigen stimulation, but only when coupled with AMPKγ2 overexpression. This combination is what we predict will improve the function of CD19-CAR T cells in our murine xenograft leukemia model and human samples.

The ethanolic extract of Korean Curcuma longa rhizome inhibits adipogenesis in 3T3-L1 adipocytes and high-fat diet-induced obese mice via activating AMPK signaling pathway

The present study aimed to evaluate the anti-obesity effect of the ethanolic extract of Korean Curcuma longa (CLE) on 3T3-L1 adipocytes and high-fat diet (HFD)-induced obesity in mouse. CLE treatment suppressed lipid accumulation and restored the differentiation-induced changes in adipogenesis- and lipolysis-related proteins by recovering phosphorylation of AMP-activated protein kinase (AMPK). Moreover, in CLE-administered HFD-induced mouse model, body weight and lipid accumulation were reduced, and the changes in factors related to adipogenesis and lipolysis were regulated in the adipose and liver tissues, confirming that these effects were due to a restoration in the phosphorylation of AMPK. In summary, these results provide important insights into the anti-obesity role of CLE by demonstrating its anti-obesity effects on adipocytes and obese mouse models, thereby suggesting the potential application of CLE as a preventive agent for obesity.

Asperosaponin VI protects alcohol-induced hepatic steatosis and injury via regulating lipid metabolism and ER stress

BackgroundAsperosaponin VI (AVI) is a natural triterpenoid saponin isolated from Dipsacus asper Wall with documented anti-inflammatory and bone protective effects. Our previous work reported that AVI protects the liver of septic mice from acute inflammatory damage. In this paper, we further explored the protective effect and the potential mechanisms of AVI in alcoholic fatty liver disease (AFLD). MethodsThe Lieber-Decarli model was constructed to evaluate the effect of AVI on AFLD in C57BL/6 J mice. Additional in vitro work was performed to investigate HepG2 cells exposed to alcohol, then analyzed the degree of liver injury by detecting the ALT and AST levels both in the liver and serum. H&E staining and Sirius red staining were used to evaluate the histopathology variations in the liver. Further, observe lipid droplets in the cytoplasm by Oil Red O staining. We detected the expression of inflammatory cytokines with qualitative PCR; ROS, MDA, SOD, and GSH-px levels were analyzed to observe oxidative stress. Finally, exploring the activation of AMPK signaling pathway by real-time PCR and Western blotting. ResultsHistological examination of liver tissue combined with serum ALT and AST levels showed a significant protective effect of AVI against alcoholic liver injury in AFLD mice. Compared with the model group, AVI evidently improved antioxidant capacity, reduced inflammatory response and lipid accumulation both in vitro and in vivo. For mechanically, it was found that AVI up-regulated phosphorylation level of AMP-activated protein kinase (AMPK) and inhibited the endoplasmic reticulum stress (ER) pathway in AFLD. ConclusionAVI protects mice from alcohol-induced hepatic steatosis and liver injury through activating AMPK signaling and repress ER stress, suggesting that it might be a potential therapeutic agent for AFLD.

AMPK Mediates Early Activation of the Unfolded Protein Response through a Positive Feedback Loop in Palmitate-Treated Muscle Cells.

Activation of the unfolded protein response (UPR) is closely related to the pathogenesis of many metabolic disorders. Accumulating evidence also shows that UPR and metabolic signaling pathways are interdependent. The AMP-activated protein kinase (AMPK) signal pathway controls the energy balance of eukaryotes. The aim of this study was therefore to investigate the possible interaction between AMPK signaling and UPR in muscle cells exposed to saturated fatty acids, as well as the potential mechanism. The saturated fatty acid palmitate was used to induce UPR in C2C12 myotubes. Compound C or knockdown of AMPKα with short hairpin RNA (shRNA) were used to inhibit the AMPK signaling pathway in palmitate-treated muscle cells. AMPK signaling in myotubes was activated using 5-amino-1-β-D-ribofuranosylimidazole-4-carboxamide (AICAR) or ex229. C2C12 myotubes were pre-treated with taurourdodeoxycholic acid (TUDCA) to inhibit UPR before adding palmitate. Real-time PCR and Western blotting were performed to evaluate the expression of UPR markers and activation of AMPK. Palmitate treatment induced UPR in C2C12 myotubes while activating AMPK signaling. Inhibition of the AMPK pathway with compound C or AMPK shRNA reduced palmitate-induced activation of UPR, while inhibition of UPR with TUDCA reduced palmitate-induced AMPK activation. This indicates a positive feedback loop between UPR and AMPK. Furthermore, activation of the AMPK pathway with AICAR or ex229 caused a dose-dependent upregulation of UPR markers, including activating transcription factor 4 (ATF4), binding immunoglobulin protein (BIP), and growth arrest and DNA damage-inducible 34 (GADD34) protein. These results provide the first evidence that AMPK signaling is involved in the early activation of UPR caused by saturated fatty acids in skeletal muscle. Furthermore, they indicate that physiological or pharmacological activation of the AMPK pathway (e.g., by exercise or phenformin, respectively) can promote muscle health and function, thereby improving the quality of life in individuals with metabolic disorders due to a high-fat diet or obesity.

Discovery of IHMT-MST1-39 as a novel MST1 kinase inhibitor and AMPK activator for the treatment of diabetes mellitus

Insulin-producing pancreatic β cell death is the fundamental cause of type 1 diabetes (T1D) and a contributing factor to type 2 diabetes (T2D). Moreover, metabolic disorder is another hallmark of T2D. Mammalian sterile 20-like kinase 1 (MST1) contributes to the progression of diabetes mellitus through apoptosis induction and acceleration of pancreatic β cell dysfunction. AMP-activated protein kinase (AMPK) is an energy sensing kinase and its activation has been suggested as a treatment option for metabolic diseases. Thus, pharmacological inhibition of MST1 and activation of AMPK simultaneously represents a promising approach for diabetes therapy. Here, we discovered a novel selective MST1 kinase inhibitor IHMT-MST1-39, which exhibits anti-apoptosis efficacy and improves the survival of pancreatic β cells under diabetogenic conditions, as well as primary pancreatic islets in an ex vivo disease model. Mechanistically, IHMT-MST1-39 activated AMPK signaling pathway in hepatocytes in vitro, combination of IHMT-MST1-39 and metformin synergistically prevented hyperglycemia and significantly ameliorated glucose tolerance and insulin resistance in diabetic mice. Taken together, IHMT-MST1-39 is a promising anti-diabetic candidate as a single agent or in combination therapy for both T1D and T2D.

Tectorigenin targets PKACα to promote GLUT4 expression in skeletal muscle and improve insulin resistance in vitro and in vivo.

The decreased expression and dysfunction of glucose transporter 4 (GLUT4), the insulin-responsive glucose transporter, are closely related to the occurrence of insulin resistance (IR). To improve the expression of GLUT4 may represent a promising strategy to prevent and treat IR and type 2 diabetes (T2DM). Here, we demonstrate that the natural compound tectorigenin (TG) enhances GLUT4 expression, glucose uptake and insulin responsiveness via activating AMP-activated protein kinase (AMPK)/myocyte enhancer factor 2 (MEF2) signaling in both normal and IR skeletal muscle cells and tissues. Accordingly, prophylactic and therapeutic uses of TG can significantly ameliorate IR and hyperglycemia in T2DM mice. Mechanistically, we identify protein kinase A catalytic subunit α (PKACα) as the target of TG to increase GLUT4 expression and TG-PKACα binding promotes the dissociation of PKACα from the regulatory subunits, leading to the activation of PKA/AMPK signaling. PKACα knockdown in local quadriceps muscles almost completely abolished the therapeutic effects of TG on IR and T2DM, as well as the enhancement on AMPK signaling and GLUT4 expression in skeletal muscle. This study supports TG as a new drug candidate to treat IR and its related diseases, but also enriches our knowledge of PKA signaling in glucose metabolism in skeletal muscle.

Semaglutide Pretreatment Induces Cardiac Autophagy to Reduce Myocardial Injury in Septic Mice.

Sepsis-induced myocardial dysfunction (SIMD) confers substantial morbidity and mortality. Semaglutide treatment has demonstrated efficacy in ameliorating sepsis-related organ damage via attenuation of inflammation, oxidative stress, and apoptotic cell death. In this study, we constructed a mouse SIMD model using cecal ligation and puncture (CLP) to explore whether semaglutide preconditioning can modulate autophagy levels and attenuate myocardial injury. C57BL/6 mice were randomly divided into six groups: sham, CLP (including CLP-6 h, CLP-12 h and CLP-24 h subgroups), semaglutide, and semaglutide+Compound-C, with five mice in each group. The latter two groups were given daily intraperitoneal injections of semaglutide for 14 days. The semaglutide+Compound-C group was given the autophagy inhibitor Compound-C intraperitoneally 1-hour before CLP surgery. After the last injection of semaglutide, SIMD mouse models were constructed by CLP surgery, while the sham group underwent a sham operation. All mice were sacrificed after surgery, and blood and myocardial specimens were collected. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of inflammatory factors and myocardial injury markers in the serum, while quantitative real-time polymerase chain reaction (qRT-PCR) and western blot was used to detect the expression of autophagic markers [microtubule-associated protein 1A/1B-light chain 3B (LC3B), Beclin-1, p62] and AMP-activated protein kinase (AMPK) in myocardial tissue. Hematoxylin and eosin (H&E) staining was used to observe pathological changes in myocardial tissue. The myocardial fibers in the sham group were normal, while those in the CLP group showed disordered arrangement, interstitial edema, and a large number of infiltrating inflammatory cells. A few vacuolar changes were observed locally in the semaglutide group, and more vacuolar changes were observed in the semaglutide+Compound-C group. Autophagy was inhibited in the CLP group mice. Compared with the CLP group, the semaglutide group showed a decreased levels of inflammatory factors (tumor necrosis factor-α, interleukin-1β) and myocardial injury markers (creatine kinase isoenzyme, cardiac troponin T) in the serum, a reduced expression of autophgic substrate p62, and an increased expression of LC3II (the lipidated form of LC3I)/LC3I (microtubule-associated protein 1A/1B-light chain 3), Beclin-1, and p-AMPK (phosphorylated AMP-activated protein kinase)/AMPK in the injured myocardial tissues of mice (p < 0.05). And the protective effects of semaglutide against SIMD were partially reversed by the treatment of AMPK inhibitor Compound-C (p < 0.05). Taken together, these data indicate that semaglutide provides protection against CLP-triggered myocardial inflammation and injury, potentially by reactivating myocardial autophagy pathways via activation of AMPK signaling. Further mechanistic studies are needed to definitively elucidate the functional significance of AMPK signaling in mediating the beneficial cardiac effects of semaglutide during sepsis.

Abstract 10414: Sesn2/AMPK Signaling Medicates Metformin's Pharmacological Action on Cardiac Mitochondrial Functional Integrity in Response to Ischemic Insults

Introduction: Metformin activates AMP-activated protein kinase (AMPK) to limit cardiac damage caused by ischemia and reperfusion (I/R). We found that a stress inducible protein Sesn2 is associated with AMPK to maintain mitochondrial oxidative phosphorylation (OXPHOS) during I/R. But the role of Sesn2/AMPK complex in the beneficial effects of metformin on ischemic insults remain unknown. Hypothesis: Metformin modulates Sesn2/AMPK signaling cascade to maintain mitochondrial integrity and respiration functions during I/R stress. Methods: Cardiomyocyte specific Sesn2 knock out (cSesn2 -/- ) and wildtype littermates (Sesn2 f/f ) C57BL/6J mice were subjected to in vivo regional ischemia of 45 min followed by injection of metformin (2 mM, i.v.) 5 min before 24 hr of reperfusion. Mitochondrial functions were determined by a Seahorse XF24 Analyzer. The sarcomere shortening and calcium transients of cardiomyocytes were measured with IonOptix Calcium and Contractility System. Results: Echocardiography data showed that I/R versus sham operations significantly reduced ejection fraction (EF) and fractional shortening (FS) in both Sesn2 f/f and cSesn2 -/- mice. Metformin treatment improved cardiac function under I/R and reduced myocardial infarct size by I/R in Sesn2 f/f but not cSesn2 -/- mice. Immunoblotting data showed metformin activated AMPK signaling and augmented mitochondrial OXPHOS Complex II levels in Sesn2 f/f but not cSesn2 -/- hearts under I/R stress conditions. Transmission electronic microscopy with immuno-gold labeling showed that Sesn2 is primarily located in the mitochondrial membrane and metformin treatment stabilized mitochondrial Sesn2 in Sesn2 f/f versus cSesn2 -/- hearts under I/R conditions. IonOptix measurements showed that metformin rescues contractile dysfunctions and impaired calcium flux by I/R in Sens2 f/f but not cSesn2 -/- cardiomyocytes. In addition, metformin treatment reduced reactive oxygen species (ROS) levels and improved mitochondrial respiration rate under I/R conditions in Sesn2 f/f but not cSesn2 -/- hearts. Conclusions: Administration of metformin benefits cardiac functions under I/R stress. Sesn2/AMPK signaling mediates mitochondrial functional integrity during I/R with metformin treatment.

Therapeutic metformin concentrations positively regulate proliferation in endometrial epithelial cells (EECs) via mTOR activation and augmented mitochondrial strength.

Metformin, an antidiabetic drug, has recently been repositioned in the treatment of several nondiabetic disorders including reproductive disorders such as polycystic ovarian syndrome (PCOS), where it improves endometrial functions. In vitro studies employing supratherapeutic concentrations (5-20 mM) of metformin, have reported anti-proliferative effects on endometrial epithelial and stromal cells. However, animal and human studies have revealed that therapeutic serum concentrations of metformin range between 20-70 µM. In the present study, the effect of therapeutic concentrations of metformin was studied on endometrial epithelial cells (EECs). Therapeutic concentrations of metformin induced proliferation in Ishikawa and HEC-1A cells. The proliferation of EECs was found mTOR dependent. Interestingly, therapeutic metformin concentrations were not able to activate the classical AMPK signaling. On the contrary, supratherapeutic metformin concentration (10 mM) inhibited mTOR and activated AMPK signaling. Microarray analysis of metformin-treated HEC-1A cells revealed dose-dependent differential effects on biological pathways associated with translation, ribosomal RNA processing, mitochondrial translation and cell proliferation. Therapeutic concentrations of metformin upregulated mitochondrial number as demonstrated by increased MitotrackerTM Red staining and enhanced succinate dehydrogenase (SDHD) expression; however higher concentration (10 mM) abrogated the same. Our results suggest that therapeutic concentrations of metformin augment mitochondrial strength and induce mTOR dependent endometrial cell proliferation.

Hepatic AMPK signaling dynamic activation in response to REDOX balance are sentinel biomarkers of exercise and antioxidant intervention to improve blood glucose control.

Antioxidant intervention is considered to inhibit reactive oxygen species (ROS) and alleviate hyperglycemia. Paradoxically, moderate exercise can produce ROS to improve diabetes. The exact redox mechanism of these two different approaches remains largely unclear. Here, by comparing exercise and antioxidant intervention on type 2 diabetic rats, we found moderate exercise upregulated compensatory antioxidant capability and reached a higher level of redox balance in the liver. In contrast, antioxidant intervention achieved a low-level redox balance by inhibiting oxidative stress. Both of these two interventions could promote glucose catabolism and inhibit gluconeogenesis through activation of hepatic AMP-activated protein kinase (AMPK) signaling; therefore, ameliorating diabetes. During exercise, different levels of ROS generated by exercise have differential regulations on the activity and expression of hepatic AMPK. Moderate exercise-derived ROS promoted hepatic AMPK glutathionylation activation. However, excessive exercise increased oxidative damage and inhibited the activity and expression of AMPK. Overall, our results illustrate that both exercise and antioxidant intervention improve blood glucose control in diabetes by promoting redox balance, despite different levels of redox state(s). These results indicate that the AMPK signaling activation, combined with oxidative damage markers, could act as sentinel biomarkers, reflecting the threshold of redox balance that is linked to effective glucose control in diabetes. These findings provide theoretical evidence for the precise management of diabetes by antioxidants and exercise.

Aspiletrein A Induces Apoptosis Cell Death via Increasing Reactive Oxygen Species Generation and AMPK Activation in Non-Small-Cell Lung Cancer Cells.

Lung cancer remains a leading cause of death in cancer patients, and deregulation of apoptosis is a serious concern in clinical practice, even though therapeutic intervention has been greatly improved. Plants are a versatile source of biologically active compounds for anticancer drug discovery, and aspiletrein A (AA) is a steroidal saponin isolated from Aspidistra letreae that has a potent cytotoxic effect on various cancer cell lines. In this study, we investigated and determined the underlying molecular mechanism by which AA induces apoptosis. AA strongly induced apoptosis in NSCLC cells by mediating ROS generation and thereby activating AMP-activated protein kinase (AMPK) signaling. Consequently, downstream signaling and levels of phosphorylated mTOR and Bcl-2 were significantly decreased. Pretreatment with either an antioxidant, N-acetylcysteine, or an AMPK inhibitor, compound C, could reverse the apoptosis-inducing effect and counteract the effect of AA on the AMPK signaling pathway. Decreased levels of Bcl-2 were due to AA-mediating Bcl-2 degradation via a ROS/AMPK/mTOR axis-dependent proteasomal mechanism. Consistently, the apoptotic-inducing effect of AA was also observed in patient-derived malignant lung cancer cells, and it suppressed an in vitro 3D-tumorigenesis. This study identified the underlying mechanism of AA on lung cancer apoptosis, thereby facilitating potential research and development of this compound for further clinical implications.

Gitogenin suppresses lung cancer progression by inducing apoptosis and autophagy initiation through the activation of AMPK signaling

Lung cancer is a leading cause of tumor-associated death worldwide. Autophagy plays a key role in regulating lung cancer progression, and is a promising option for lung cancer treatment. Saponins are a group of naturally occurring plant glycosides, characterized by their strong foam-forming properties in aqueous solution, and exert various biological properties, such as anti-inflammation and anti-cancer. In the present study, we for the first time explored the effects of gitogenin (GIT), an important saponin derived from Tribulus longipetalus, on lung cancer progression both in vitro and in vivo. We found that GIT markedly reduced the proliferation and induced apoptosis in lung cancer cells through increasing the cleavage of Caspase-3 and poly (ADP-ribose) polymerases (PARPs). In addition, GIT-incubated lung cancer cells exhibited clear accumulation of autophagosome, which was essential for GIT-suppressed lung cancer. Mechanistically, GIT-induced autophagy initiation was mainly through activating AMP-activated protein kinase (AMPK) and blocking protein kinase B (AKT) signaling pathways, respectively. Moreover, the autophagic flux was disrupted in GIT-treated lung cancer cells, contributing to the accumulation of impaired autophagolysosomes. Importantly, we found that suppressing autophagy initiation could abolish GIT-induced cell death; however, autophagosomes accumulation sensitized lung cancer cells to cell death upon GIT treatment. More in vitro experiments showed that GIT led to reactive oxygen species (ROS) production in lung cancer cells, which was also involved in the modulation of apoptosis. The in vivo findings confirmed the effects of GIT against lung cancer progression with undetectable toxicity to organs. In conclusion, we provided new insights into the treatment of lung cancer, and GIT might be an effective strategy for future clinical application.

Epithelial-Mesenchymal Transition Suppresses AMPK and Sensitizes Cancer Cells to Pyroptosis under Energy Stress.

Epithelial–mesenchymal transition (EMT) is implicated in tumor metastasis and therapeutic resistance. It remains a challenge to target cancer cells that have undergone EMT. The Snail family of key EMT-inducing transcription factors directly binds to and transcriptionally represses not only epithelial genes but also a myriad of additional genomic targets that may carry out significant biological functions. Therefore, we reasoned that EMT inherently causes various concomitant phenotypes, some of which may create targetable vulnerabilities for cancer treatment. In the present study, we found that Snail transcription factors bind to the promoters of multiple genes encoding subunits of the AMP-activated protein kinase (AMPK) complex, and expression of AMPK genes was markedly downregulated by EMT. Accordingly, high AMPK expression in tumors correlated with epithelial cell markers and low AMPK expression in tumors was strongly associated with adverse prognosis. AMPK is the principal sensor of cellular energy status. In response to energy stress, AMPK is activated and critically reprograms cellular metabolism to restore energy homeostasis and maintain cell survival. We showed that activation of AMPK by energy stress was severely impaired by EMT. Consequently, EMT cancer cells became hypersensitive to a variety of energy stress conditions and primarily underwent pyroptosis, a regulated form of necrotic cell death. Collectively, the study suggests that EMT impedes the activation of AMPK signaling induced by energy stress and sensitizes cancer cells to pyroptotic cell death under energy stress conditions. Therefore, while EMT promotes malignant progression, it concurrently induces collateral vulnerabilities that may be therapeutically exploited.