AMPK Phosphorylation Research Articles

A novel blood-based bioassay to monitor adiponectin signaling

The diverse beneficial effects of adiponectin-receptor signaling, including its impact on the regulation of inflammatory processes in vivo, have resulted in development of adiponectin receptor agonists as a treatment for metabolic disorders. However, there are no established non-invasive bioassays for detection of adiponectin target engagement in humans or animal models. Here, we designed an assay using small amounts of blood to assess adiponectin action. Specifically, we tested effects of the small 10-amino acid peptide adiponectin receptor agonist, ALY688, in a sublethal LPS endotoxemia model in mice. LPS-induced pro-inflammatory cytokine levels in serum were significantly reduced in mice treated with ALY688, assessed via multiplex ELISA in flow cytometry. Furthermore, ALY688 alone significantly induced TGF-β release in serum 1 h after treatment and was elevated for up to 24 h. Additionally, using a flow-cytometry panel for detection of changes in circulating immune cell phenotypes, we observed a significant increase in absolute T cell counts in mice after ALY688 treatment. To assess changes in intracellular signaling effectors downstream of adiponectin, phospho-flow cytometry was conducted. There was a significant increase in phosphorylation of AMPK and p38-MAPK in mice after ALY688 treatment. We then used human donor immune cells (PBMCs) treated with ALY688 ex vivo and observed elevation of AMPK and p38-MAPK phosphorylation from baseline in response to ALY688. Together, these results indicate we can detect adiponectin action on immune cells in vivo by assessing adiponectin signaling pathway for AMPK and p38-MAPK, as well as pro-inflammatory cytokine levels. This new approach provides a blood-based bioassay for screening adiponectin action.

Folate induces stemness and increases oxygen consumption under glucose deprivation by notch-1 pathway activation in colorectal cancer cell.

Evidence for folate's protective effects on neural tube defects led the USA and Chile to start mandatory folic acid (FA) fortification programs, decreasing up to 50%. However, ∼30% of the population consuming fortified foods reach supraphysiologic serum levels. Although controversial, several epidemiological and clinical observations suggest that folate increases cancer risk, giving concern about the risks of FA supplementation. The Cancer stem cells (CSCs) model has been used to explain survival to anticancer therapies. The Notch-1 pathway plays a role in several cancers and is associated with the stemness process. Different studies show that modulation of metabolic pathways regulates stemness capacity in cancer. Supraphysiologic concentrations of FA increase the proliferation of HT-29 cells by Notch-1 activation. However, whether folate can induce a stemness-like phenotype in cancer is not known. We hypothesized that FA protects from glucose deprivation-induced cell death through Notch-1 activation. HT-29 cells were challenged with glucose deprivation at basal (20 nM) and supraphysiological (400 nM) FA and 5-MTHF concentrations. We analyzed changes in stemness-like gene expression, cell death and different energetic metabolic functions. Supraphysiological concentrations of FA increased stemness-like genes, and improved survival and oxygen consumption, inducing AMPK phosphorylation and HSP-70 protein expression. We evaluated the Notch-1 pathway using the DAPT and siRNA as inhibitors, decreasing the stemness-like gene expression and preventing the FA protection against glucose deprivation-induced cell death. Moreover, they decreased oxygen consumption and AMPK phosphorylation. These results suggest that FA protects against glucose deprivation. These effects were associated with AMPK activation, a critical metabolic mediator in nutrient consumption and availability that activates the Notch-1 pathway.

Abstract 7059: Remodeling of anaplastic thyroid cancer metabolic signature by natural alkaloid berberine

Abstract Anaplastic thyroid cancer (ATC) is a rare, fatal cancer with a five-year survival of 4%. Universally diagnosed at stage IV, ATC is characterized by its lack of differentiation and metabolic dysregulation. Refractory to all established therapies, we propose natural alkaloid berberine (BBR) as a therapeutic with multitarget efficacy to alter mitochondrial metabolism and reprogram ATC’s aggressive phenotype. Assays used monocyte cell line U937, ATC cell lines T238 and SW1736, and immortalized normal thyroid cell line Nthy-ori-3-1. All assays used 100µM concentration of BBR or DMSO vehicle control for 24 hours for a tumoricidal effect. Validation of in vitro findings via RNA-Seq was conducted by Genewiz from Azenta and Qiagen’s Ingenuity Pathway Analysis was used for in silico modeling. Mitochondria were isolated via differential centrifugation. Modeling the ATC tumor microenvironment, U937 cells were activated and polarized into a proinflammatory macrophage phenotype. BBR treatment at the activation/polarization stages, 33 soluble inflammatory mediators were downregulated in the conditioned media compared to controls. In targeting the aggressiveness of anaplastic thyroid disease, BBR slowed proliferation by 80% selectively in ATC cells from 48 to 72 hours, while sparing normal cells. BBR reduced migratory capacity by 30% in ATC cells after 24, 48, and 72 hours and reduced migration and invasion by 30%. These observations were substantiated by Western blot analysis - BBR selectively decreased phosphorylation of MEK, ERK, and ribosomal protein S6, crucial downstream regulators of the pro-proliferative and pro-survival pathways in ATC cells. Further, BBR specifically modulated cancer-associated metabolism as observed through an increase in AMPKα phosphorylation, a major rate-limiting protein in cancer-induced dysregulation with an anti-tumor effect. Validation of in vitro findings via RNA-Seq revealed more than 400 significantly differentially expressed genes involved in mitochondrial metabolism, glycometabolism, sirtuin signaling, apoptosis, and proliferation. Following a comprehensive analysis, we identified significant downregulation of 25 of 37 total mitochondrially encoded genes (MTs) and 13 of 13 mitochondrially encoded protein-coding genes comprising the oxidative phosphorylation complexes illuminating a clear link between BBR treatment and altered mitochondrial metabolism in ATC. Additionally, protein expression of significantly downregulated mitochondrial genes identified via RNA Seq was validated via Western blot demonstrating decreased mitochondrially-encoded protein expression related to oxidative phosphorylation. This work reveals a novel role for BBR as an inhibitor of mitochondrial metabolism that can be used to reprogram the aggressive nature of ATC and open the door for promising combination therapy in the treatment of ATC. Citation Format: Tara Jarboe, Nicole R. DeSouza, Kaci Kopec, Jan Geliebter, Xiu-Min Li, Raj K. Tiwari. Remodeling of anaplastic thyroid cancer metabolic signature by natural alkaloid berberine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7059.

Abstract 4549: Combinations of ciclopirox olamine and metformin exert synergistic antitumor activity in triple-negative breast cancer

Abstract Introduction: Triple-negative breast cancer (TNBC) represents a challenging subtype of breast cancer because of its limited treatment options. Recent research has highlighted the potential benefits of combining pharmacological agents to enhance therapeutic outcomes. Studies show that Ciclopirox olamine (CPX), an off-patent anti-fungal drug, induces cell cycle G1-phase arrest and activates the intrinsic apoptosis pathway by downregulating the anti-apoptotic proteins. Meanwhile, metformin, a safe and first-line drug for the treatment of type II diabetes, has been shown to induce cell cycle S-phase arrest and activates the extrinsic apoptosis pathway via induction of endogenous TRAIL in TNBC cells. However, whether the combinations of CPX and metformin would exert potent antitumor activity against TNBC remains unclear. Methods: Cell proliferation (MTS) assays and LIVE/DEAD cell imaging were used to determine the combined inhibitory effects on TNBC cells. IC50s and the combination index (CI) were calculated. Flow cytometry assays were carried out to analyze if the combinations are more potent to block cell cycle progression. Apoptotic-specific ELISA and flow cytometry analysis of Annexin V-PI staining were performed to assess if the combinations are significantly more effective to induce apoptosis. Western blot assays were used to examine if the combinations markedly induced the expression of TRAIL and cleavages of PARP and caspases. Tumor xenograft models will be established to test the in vivo antitumor activity of CPX and metformin either alone or in combinations. Results: Data indicated that both CPX and metformin independently inhibited the proliferation of TNBC cells. A synergistic effect was observed when CPX and metformin were used in combination, resulting in a more potent inhibition of TNBC cell proliferation. The combinatorial treatment effectively suppressed colony formation of TNBC cells. Synergy between CPX and metformin was partially linked to the enhanced activation of the AMPK-mTOR pathway, as the combination significantly enhanced the phosphorylation of AMPKα. The combinations also induced enhanced apoptosis in TNBC cells through both the intrinsic and extrinsic pathways. The combinatorial effects of CPX and metformin on in vivo tumor growth are under investigation. Conclusions: Combinations of CPX and metformin show synergistic effects on inhibiting TNBC cell proliferation. Our studies suggest a potential therapeutic strategy for TNBC, with the combinations of these agents exhibiting an enhanced antitumor activity. Further investigation is warranted to validate the results and explore their clinical applications. Key words: Triple-negative breast cancer, Ciclopirox olamine, Metformin, Combination, Apoptosis Citation Format: Yakun Wu, Hui Lyu, Congcong Tan, Margaret E. Larsen, Shile Huang, Bolin Liu. Combinations of ciclopirox olamine and metformin exert synergistic antitumor activity in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4549.

Abstract 5937: KS20226, a first-in-class IRP2 inhibitor, preferentially induces reprogramming of iron metabolism and suppresses tumor growth in colorectal cancer

Abstract Background: Iron homeostasis plays a role in redox activity, mitochondrial function, and cell cycle regulation. Specifically, the dysregulation of iron metabolism is implicated in malignant transformation and cancer progression. Iron regulatory protein 2 (IRP2) is crucial for maintaining balance within iron levels and primarily controls genes associated with iron utilization. Our discovery of KS20226, a first-in-class IRP2 inhibitor, reveals remarkable potential as an effective anti-cancer drug candidate. We have elucidated the mechanisms underlying the therapeutic effects of KS20226, thereby highlighting the feasibility of targeting IRP2 in colorectal cancer (CRC). Methods: The proliferation inhibitory activity of KS20226 was assessed the CCK-8 and 3D Spheroid models. To investigate mitochondrial function, we conducted measurement of oxidative phosphorylation (OCR) and glycoPER. Furthermore, gene expression analysis was performed through RNA sequencing (RNA-seq) using the Illumina HiSeq 2500 system platform (Macrogen Inc.). Differential expressed gene (DEG) analysis was carried out using the R package DESeq2, and pathway enrichment analysis was analyzed using GSEA (v_4.2.3.). For the observation of autophagic morphology, we utilized transmission electron microscopy (TEM), immunofluorescence staining targeting the autophagy marker LC3B, and immunoblotting. Results: KS20226 exhibited a remarkable growth inhibitory effect in 10 different CRC cell lines, with GI50 values ranging from 1 to 10 μM. KS20226 suppressed IRP2 expression and the corresponding occupancy of the iron-responsive elements of ferritin H and transferrin receptor 1, resulting in iron deprivation, and efficiently controlled CRC growth. Pharmacologic inhibition of IRP2 induced reprogramming of mitochondrial respiration, reducing reliance on oxidative phosphorylation. The observed mitochondrial dysfunction induced by KS20226 was correlated with pathway analysis, revealing a decrease in the gene-set for HALLMARK_OXIDATIVE_PHOSPHORYLATION (NES = -1.38, p-value = 0.008) and an increase in the gene-set for POSITIVE_REGULATION_OF_AUTOPHAGY (NES=1.43, p-value=0.016). Immunoblotting demonstrated that KS20226 activated the phosphorylation of AMPK, ULK, and Beclin1 cascade, leading to the autophagic cell death of the CRC cells. Conclusions: The reliance of cancer cells on iron metabolism to drive proliferation has led to the development of therapeutic strategies targeting the inhibition of IRP2. KS20226, a first-in-class IPR2 inhibitor, exhibits distinct therapeutic properties, such as perturbation of mitochondrial iron metabolism and induction of autophagic cell death. Our findings represent important progress toward exploiting IRP2-mediated abnormal iron metabolism in cancer, an unexplored area of research with substantial unmet needs. Citation Format: Jieon Hwang, Areum Park, Chinwoo Kim, Chang Gon Kim, Jaesung Kwak, Byungil Kim, Hyunjin Shin, Minhee Ku, Jaemoon Yang, Ayoung Baek, Jiwon Choi, Hocheol Lim, Kyoung Tai No, Xianghua Zhao, Uyeong Choi, Tae Il Kim, Kyu-Sung Jeong, Hyuk Lee, Sang Joon Shin. KS20226, a first-in-class IRP2 inhibitor, preferentially induces reprogramming of iron metabolism and suppresses tumor growth in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5937.

Abstract 1569: Keratin X promotes cell metastasis and sorafenib resistance via the AMPKα/Snail axis in hepatocellular carcinoma

Abstract A great majority of liver cancers are developed from cirrhotic livers. The key molecules involved in cirrhosis-mediated hepatocarcinogenesis have not been clearly defined. Understanding the oncogenic pathways and their associations with cancer growth, metastasis and drug resistance may help scientists devise new effective therapeutic agents. In the present study, we identified the intermediate filament-forming protein keratin X (KRTX) as a key factor in cirrhosis-mediated hepatocarcinogenesis. KRTX was highly expressed in HCC tissues and correlated with poor survival outcomes. Experimental verification revealed that overexpression of KRTX enhanced cell growth, tumor cell metastasis, glycolysis and sorafenib resistance. Conversely, knockdown of KRTX led to opposite effects. Mechanistically, KRTX inhibited AMPKα phosphorylation, thereby enhancing Snail protein stability. Additionally, TGFβ1 positively regulated KRTX expression, resulting in a KRTX/AMPKα (Thr172)/Snail axis-mediated increase in cell motility. Treating with 2-deoxyglucose reversed the KRTX-induced metastasis and sorafenib resistance. In clinic, KRTX expression is positively correlated with Snail overexpression and high KRTX/Snail expression had poor prognosis in HCC patients. Our data revealed a signaling pathway from TGFβ1, KRTX, through p-AMPKα (Thr172) to Snail. Targeting KRTX and its pathway may be effective for treating patients with metastatic HCC. Citation Format: Yang-Hsiang Lin, Chau-Ting Yeh. Keratin X promotes cell metastasis and sorafenib resistance via the AMPKα/Snail axis in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1569.

Abstract 599: Combination of CDK4/6 inhibitors with biguanides for treatment of triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer (BC) and is associated with early relapse and high frequency of metastasis. This has been attributed in part to the lack of targeted therapies. TNBC occurs more frequently in younger women of African and Hispanic ancestry and with a higher risk of mortality than women of European ancestry. Emerging data shows activation of the AKT/mTOR pathway by insulin may occur in aggressive TNBC and inhibition of mTOR signaling by rapamycin, a potent mTOR inhibitor, induces cell cycle arrest and downregulation of cyclin D1 in TNBC cells. CDK4/6 inhibitors have demonstrated to be an effective therapeutic for estrogen receptor positive BC and although their use in TNBC patients remains uncertain their use in combination with other targeted therapies may be promising. We have developed a set of newly designed biguanides with potent anticancer action and safety doses in vivo that in combination with CDK4/6 inhibitors block TNBC cell growth in vitro and in vivo. Using TNBC cell proliferation in vitro assays, new biguanides show dose-dependent inhibition of cell proliferation (P<0.01). Further, combination of biguanides with ribocliclib, an orally bioavailable CDK4/6 inhibitor, produced additive and synergistic effects in TNBC cell lines in vitro (P<0.01). Biguanides are known to activate de LKB1-AMPK pathway. Western immunoblots showed our biguanide analogues also induce AMPK phosphorylation and significantly reduce phosphorylation of downstream mTOR signaling pathway components including S6 ribosomal protein and 4E-BP1, an effect that is potentiated by combination treatment with ribociblib. Further, cyclin D1 is known to be overexpressed in about 50% of BCs and binding to CDK 4/6 induces phosphorylation of Rb and cell cycle progression. Treatment with biguanides downregulated cyclin D1 expression in TNBC cells and combination treatment of ribociclib with biguanide JD006 was more effective at decreasing Rb phosphorylation than each drug alone. In addition, seahorse analysis evaluated mitochondrial respiratory function. Treatment with biguanides inhibited oxidative phosphorylation in a dose dependent manner, an important metabolic pathway that can drive drug resistance to therapy in cancer cells. In vivo, combination treatment of ribociclib with biguanide JD006 was more effective than each agent alone in stopping human TNBC xenograft progression in nude mouse models (P<0.05). Our preliminary data offer evidence that human TNBC are highly vulnerable to metabolic reprogramming by new biguanides. This work can yield insight on unanticipated mechanisms of biguanide action and rationale for new combination therapies with CDK 4/6 inhibitors as a new option for treatment of TNBC. Funding: CBCRP B27IB3869, JCCC Breast Cancer Award, Hickey Family Foundation, NCI U54 CA143930, Team Research Grant, UCLA TDG. Citation Format: Mario Morales Martinez, Eduardo Mauricio Gonzalez, Gang Deng, Gaoyuan Ma, Hyunsoo Kim, Linsey Stiles, Nalo Hamilton, Michael E. Jung, Richard J. Pietras, Diana C. Marquez-Garban. Combination of CDK4/6 inhibitors with biguanides for treatment of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 599.

The GPR40 novel agonist SZZ15-11 improves non-alcoholic fatty liver disease by activating the AMPK pathway and restores metabolic homeostasis in diet-induced obese mice.

Non-alcoholic fatty liver is the most common cause of chronic liver disease. GPR40 is a potential therapeutic target for energy metabolic disorders. GPR40 is a potential therapeutic target for energy metabolic disorders. SZZ15-11 is a newly synthesized GPR40 agonist. In this study, we estimate the potency of SZZ15-11 in fatty liver treatment. In vivo, diet-induced obese (DIO) mice received SZZ15-11 (50 mg/kg) and TAK875 (50 mg/kg) for 6 weeks. Blood glucose and lipid, hepatocyte lipid and liver morphology were analysed. In vitro, HepG2 cells and GPR40-knockdown HepG2 cells induced with 0.3 mM oleic acid were treated with SZZ15-11. Triglyceride and total cholesterol of cells were measured. At the same time, the AMPK pathway regulating triglycerides and cholesterol esters synthesis was investigated via western blot and quantitative polymerase chain reaction in both liver tissue and HepG2 cells. SZZ15-11 was found to not only attenuate hyperglycaemia and hyperlipidaemia but also ameliorate fatty liver disease in DIO mice. At the same time, SZZ15-11 decreased triglyceride and total cholesterol content in HepG2 cells. Whether examined in the liver of DIO mice or in HepG2 cells, SZZ15-11 upregulated AMPKα phosphorylation and then downregulated the expression of the cholesterogenic key enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase and inhibited acetyl-CoA carboxylase activity. Furthermore, SZZ15-11 promotes AMPK activity via [cAMP]i accumulation. This study confirmed that SZZ15-11, a novel GPR40 agonist, improves hyperlipidaemia and fatty liver, partially via Gs signalling and the AMPK pathway in hepatocytes.

Screening of Platycodonis Radix Fractions for Antiobesity Activities and Elucidation of Its Molecular Mechanisms in High-Fat Diet-Fed C57BL/6 Mice.

Obesity is a threat to public health and effective new medications are required. Platycodonis Radix (PR) is a traditional medicinal/dietary plant with activities against obesity. Using mice given a diet rich in fat, the antiobesity components of PR were identified and their molecular mechanisms were clarified further in this investigation. Initially, the impacts of PR fractions on liver histology and biochemical markers were assessed. Subsequently, the degrees of lipogenic and lipolytic gene and protein expressions were determined. Oral administration of PR polysaccharides (PG) (0.80 g/kg body weight) improved liver function (alanine aminotransferase and aspartate aminotransferase) and its antioxidant activities (total superoxide dismutase, glutathione peroxidase, and malondialdehyde), as well as alleviated blood lipid (total cholesterol, total triglyceride, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol) values, inflammatory systemic (TNF-α and IL-1β), and histological abnormalities within the liver. Furthermore, PG administration downregulated the expression for lipogenic genes (ACC and FAS) and upregulated the expression for the lipolytic gene (PPARα, LPL, CPT1, and HSL). Importantly, PG raised AMPK phosphorylation and decreased SREBP-1c protein synthesis. Thus, it is possible that PG stimulates the AMPK-LPL/HSL path (lipolytic route) plus the AMPK-ACC/PPARα-CPT1 path (associated to β-oxidation of fatty acids), while inhibiting the AMPK/(SREBP-1c)-ACC/FAS path (lipogenic route). In summary, PG has the ability to regulate lipid metabolism, and it may be useful to pharmacologically activate AMPK with PG to prevent and cure obesity.

Glutamine suppresses senescence and promotes autophagy through glycolysis inhibition-mediated AMPKα lactylation in intervertebral disc degeneration

Regulating metabolic disorders has become a promising focus in treating intervertebral disc degeneration (IDD). A few drugs regulating metabolism, such as atorvastatin, metformin, and melatonin, show positive effects in treating IDD. Glutamine participates in multiple metabolic processes, including glutaminolysis and glycolysis; however, its impact on IDD is unclear. The current study reveals that glutamine levels are decreased in severely degenerated human nucleus pulposus (NP) tissues and aging Sprague-Dawley (SD) rat nucleus pulposus tissues, while lactate accumulation and lactylation are increased. Supplementary glutamine suppresses glycolysis and reduces lactate production, which downregulates adenosine-5’-monophosphate-activated protein kinase α (AMPKα) lactylation and upregulates AMPKα phosphorylation. Moreover, glutamine treatment reduces NP cell senescence and enhances autophagy and matrix synthesis via inhibition of glycolysis and AMPK lactylation, and glycolysis inhibition suppresses lactylation. Our results indicate that glutamine could prevent IDD by glycolysis inhibition-decreased AMPKα lactylation, which promotes autophagy and suppresses NP cell senescence.

The regulation of AMPK pathway in liver abnormal lipid deposition caused by high carbohydrate diet in rice field eel

AMP-activated protein kinase-alpha 1 (AMPKα1) plays a vital role in glucose and lipid metabolism. Five isonitrogenous and isolipid diets with starch levels (15%, 20%, 25%, 30% and 35%) were formulated to study the modulation of AMPKα1 in hepatic lipid deposition induced by high gelatinized starch diet in rice field eel (Monopterus albus) (initial body weight, 30.00 ± 0.10 g) and the growth trial lasted for 8 weeks. The results showed that dietary gelatinized starch did not affect the growth performance of rice filed eel compared with S15 group. High gelatinized starch could induce AMPKα1 expression and glucose catabolism, and inhibit hepatic glycogen synthesis without effecting glucose content. Dietary ≥ 30% gelatinized starch began to dramatically suppress gluconeogenesis and promote the lipid synthesis, followed by a higher triglyceride (TG) and hepatic crude lipid. Dietary 20–30% gelatinized starch induced phosphorylation of AMPKα compared to 15% gelatinized starch diet, whereas 35% starch dramatically suppressed AMPKα phosphorylation together with unnormal increase in phosphorylation of forkhead box O1 A (FOXO1A) and acetyl-CoA carboxylase (ACC), TG content compared to 30% starch. In short, high gelatinized starch could induce AMPKα1 expression and promoted systemic metabolism, whereas dietary ≥ 25% gelatinized starch initially suppressed AMPKα1 activation and caused abnormal lipid deposition in liver of rice field eel.

Decreased AdipoR1 signaling and its implications for obesity-induced male infertility

Obesity is among the risk factors for male infertility. Although several mechanisms underlying obesity-induced male subfertility have been reported, the entire mechanism of obesity-induced male infertility still remains unclear. Here, we show that sperm count, sperm motility and sperm fertilizing ability were decreased in male mice fed a high-fat diet and that the expression of the AdipoR1 gene and protein was decreased, and the expression of pro-apoptotic genes and protein increased, in the testis from mice fed a high-fat diet. Moreover, we demonstrate that testes weight, sperm count, sperm motility and sperm fertilizing ability were significantly decreased in AdipoR1 knockout mice compared to those in wild-type mice; furthermore, the phosphorylation of AMPK was decreased, and the expression of pro-apoptotic genes and proteins, caspase-6 activity and pathologically apoptotic seminiferous tubules were increased, in the testis from AdipoR1 knockout mice. Furthermore, study findings show that orally administrated AdipoRon decreased caspase-6 activity and apoptotic seminiferous tubules in the testis, thus ameliorating sperm motility in male mice fed a high-fat diet. This was the first study to demonstrate that decreased AdipoR1/AMPK signaling led to increased caspase-6 activity/increased apoptosis in the testis thus likely accounting for male infertility.

LKB1 regulates autophagy through AMPK/TOR signaling pathway to alleviate the damage caused by Vibrio alginolyticus infection

LKB1 (liver kinase B1) is a key upstream kinase of AMPK and plays an important role in various cellular activities. While the function and mechanism of LKB1 have been widely reported in the study of tumor, there are few reports on its role in bacterial infectious diseases, especially in shrimp. In the present study, molecular characterization revealed that LvLKB1 has an open reading frame (ORF) of 1266 bp encoding 421 amino acids with a molecular weight of about 48 KDa, including the kinase region, N-terminal regulatory domain and C-terminal regulatory domain. LvLKB1 in hepatopancreas and hemocytes was significantly upregulated after infection with Vibrio alginolyticus (V. alginolyticus). After silencing LvLKB1 gene in Litopenaeus vannamei (L. vannamei) and artificially infecting V. alginolyticus, the survival rate of L. vannamei was significantly decreased. Subsequently, it was found that the expression of inflammatory factors in hepatopancreas and hemocytes of shrimp was up-regulated, and the expression of lipid oxidation factors was decreased after silencing LKB1, leading to the phenomenon of lipid accumulation in hepatopancreas. In order to explore the mechanism, autophagy levels of shrimp were detected after silencing LKB1, which showed that autophagy levels in hepatopancreas and hemocytes were significantly reduced. Further studies conclusively showed that silencing LvLKB1 inhibited AMPK phosphorylation induced by V. alginolyticus infection, thereby activating TOR pathway and inhibiting autophagy in shrimp. These results indicate that LvLKB1 regulates autophagy through AMPK/TOR signaling pathway to alleviate the damage caused by V. alginolyticus infection.

Exercise Improves Heart Function after Myocardial Infarction: The Merits of AMPK.

AMPK is considered an important protein signaling pathway that has been shown to exert prominent cardioprotective effects on the pathophysiological mechanisms of numerous diseases. Following myocardial infarction, severe impairment of cardiac function occurs, leading to complications such as heart failure and arrhythmia. Therefore, protecting the heart and improving cardiac function are important therapeutic goals after myocardial infarction. Currently, there is substantial ongoing research on exercise-centered rehabilitation training, positioning exercise training as a significant nonpharmacological approach for preventing and treating numerous cardiovascular diseases. Previous studies have reported that exercise can activate AMPK phosphorylation and upregulate the AMPK signaling pathway to play a cardioprotective role in coronary artery disease, but the specific mechanism involved remains to be elucidated. This review discusses the role and mechanism of the exercise-mediated AMPK pathway in improving postinfarction cardiac function through existing studies and describes the mechanism of exercise-induced myocardial repair of AMPK from multiple perspectives to formulate a reasonable and optimal exercise rehabilitation program for the prevention and treatment of myocardial infarction patients in the clinic.

Ectopic OR1A1 activation ameliorates hepatic lipid deposition through AMPK/SREBP-1/FASN pathway by three monoterpenes

Ectopic olfactory receptors, such as OR1A1, are involved in the regulation of lipid metabolism homeostasis. In this study, we investigated the effects of three monoterpenes, (+)-limonene, (−)-carvone and thymol, upon hepatic lipid deposition. Oral administration of (+)-limonene, (−)-carvone and thymol for 8 weeks in high-fat-diet (HFD)-fed mice significantly lowered the levels of fasting blood glucose, improved glucose intolerance, serum lipid parameters, adiposity, and hepatic steatosis. In LO2 cells, (+)-limonene, (−)-carvone and thymol increased the cyclic adenosine monophosphate (cAMP), induced protein kinase A (PKA) activity and phosphorylation of AMPK, downregulated the expression of SREBP-1 and FASN, and eventually led to a reduction in hepatic lipid deposition. Meanwhile, these beneficial effects in cultured LO2 cells were negated by OR1A1 knockdown and the adenylyl cyclase inhibitor SQ22536, respectively, indicating that they can ameliorate disorders of lipid metabolism through activating OR1A1 and subsequent AMPK/SREBP-1/FASN pathway, indicating OR1A1 as a potential target for counteracting hepatic steatosis.

The effects of restraint stress on ceramide metabolism disorders in the rat liver: the role of CerS6 in hepatocyte injury

BackgroundStress is implicated in various pathological conditions leading to liver injury. Existing evidence suggests that excessive stress can induce mitochondrial damage in hepatocytes, yet the underlying mechanism remains unclear. Ceramide synthase 6 (CerS6)-derived C16:0 ceramide is recognised as a lipotoxic substance capable of causing mitochondrial damage. However, the role of CerS6 in stress has received insufficient attention. This study aimed to explore the involvement of CerS6 in stress-induced hepatic damage and its associated mechanisms.MethodsThe rat restraint stress model and a corticosterone (CORT)-induced hepatocyte stress model were employed for in vivo and in vitro experimental analyses, respectively. Changes in mitochondrial damage and ceramide metabolism in hepatocytes induced by stress were evaluated. The impact of CORT on mitochondrial damage and ceramide metabolism in hepatocytes was assessed following CerS6 knockdown. Mitochondria were isolated using a commercial kit, and ceramides in liver tissue and hepatocytes were detected by LC–MS/MS.ResultsIn comparison to the control group, rats subjected to one week of restraint exhibited elevated serum CORT levels. The liver displayed significant signs of mitochondrial damage, accompanied by increased CerS6 and mitochondrial C16:0 ceramide, along with activation of the AMPK/p38 MAPK pathway. In vitro studies demonstrated that CORT treatment of hepatocytes resulted in mitochondrial damage, concomitant with elevated CerS6 and mitochondrial C16:0 ceramide. Furthermore, CORT induced sequential phosphorylation of AMPK and p38 MAPK proteins, and inhibition of the p38 MAPK pathway using SB203580 mitigated the CORT-induced elevation in CerS6 protein. Knocking down CerS6 in hepatocytes inhibited both the increase in C16:0 ceramide and the release of mitochondrial cytochrome c induced by CORT.ConclusionsCerS6-associated C16:0 ceramide plays a mediating role in stress-induced mitochondrial damage in hepatocytes. The molecular mechanism is linked to CORT-induced activation of the AMPK/p38 MAPK pathway, leading to upregulated CerS6.

Regulation of NCOA4-mediated iron recycling ameliorates paraquat-induced lung injury by inhibiting ferroptosis

Paraquat (PQ) is an irreplaceable insecticide in many countries for the advantage of fast-acting and broad-spectrum. However, PQ was classified as the most prevailing poisoning substance for suicide with no specific antidote. Therefore, it is imperative to develop more effective therapeutic agents for the treatment of PQ poisoning. In the present study, both the RNA-Seq and the application of various cell death inhibitors reflected that ferroptosis exerts a crucial regulatory role in PQ poisoning. Moreover, we found PQ strengthens lipid peroxidation as evidenced by different experimental approaches. Of note, pretreatment of iron chelation agent DFO could ameliorate the ferroptotic cell death and alleviate the ferroptosis-related events. Mechanistically, PQ treatment intensively impaired mitochondrial homeostasis, enhanced phosphorylation of AMPK, accelerated the autophagy flux and triggered the activation of Nuclear receptor coactivator 4-ferritin heavy chain (NCOA4-FTH) axis. Importantly, the activation of autophagy was observed prior to the degradation of ferritin, and inhibition of autophagy could inhibit the accumulation of iron caused by the ferritinophagy process. Genetic and pharmacological inhibition of ferritinophagy could alleviate the lethal oxidative events, and rescue the ferroptotic cell death. Excitingly, in the mouse models of PQ poisoning, both the administration of DFO and adeno-associated virus-mediated FTH overexpression significantly reduced PQ-induced ferroptosis and improved the pathological characteristics of pulmonary fibrosis. In summary, the current work provides an in-depth study on the mechanism of PQ intoxication, describes a framework for the further understanding of ferroptosis in PQ-associated biological processes, and demonstrates modulation of iron metabolism may act as a promising therapeutic agent for the management of PQ toxicity.Graphical

Mito-metformin protects against mitochondrial dysfunction and dopaminergic neuronal degeneration by activating upstream PKD1 signaling in cell culture and MitoPark animal models of Parkinson's disease.

Impaired mitochondrial function and biogenesis have strongly been implicated in the pathogenesis of Parkinson's disease (PD). Thus, identifying the key signaling mechanisms regulating mitochondrial biogenesis is crucial to developing new treatment strategies for PD. We previously reported that protein kinase D1 (PKD1) activation protects against neuronal cell death in PD models by regulating mitochondrial biogenesis. To further harness the translational drug discovery potential of targeting PKD1-mediated neuroprotective signaling, we synthesized mito-metformin (Mito-Met), a mitochondria-targeted analog derived from conjugating the anti-diabetic drug metformin with a triphenylphosphonium functional group, and then evaluated the preclinical efficacy of Mito-Met in cell culture and MitoPark animal models of PD. Mito-Met (100-300 nM) significantly activated PKD1 phosphorylation, as well as downstream Akt and AMPKα phosphorylation, more potently than metformin, in N27 dopaminergic neuronal cells. Furthermore, treatment with Mito-Met upregulated the mRNA and protein expression of mitochondrial transcription factor A (TFAM) implying that Mito-Met can promote mitochondrial biogenesis. Interestingly, Mito-Met significantly increased mitochondrial bioenergetics capacity in N27 dopaminergic cells. Mito-Met also reduced mitochondrial fragmentation induced by the Parkinsonian neurotoxicant MPP+ in N27 cells and protected against MPP+-induced TH-positive neurite loss in primary neurons. More importantly, Mito-Met treatment (10 mg/kg, oral gavage for 8 week) significantly improved motor deficits and reduced striatal dopamine depletion in MitoPark mice. Taken together, our results demonstrate that Mito-Met possesses profound neuroprotective effects in both in vitro and in vivo models of PD, suggesting that pharmacological activation of PKD1 signaling could be a novel neuroprotective translational strategy in PD and other related neurocognitive diseases.

Colchicine alleviates myocardial ischemia-reperfusion injury in mice by activating AMPK

To investigate the protective effect of colchicine against myocardial ischemia-reperfusion injury (I/R) and explore the underlying mechanism. H9C2 cells exposed to hypoxia/reoxygenation (H/R) were treated with 3 nmol/L colchicine, after which the changes in cell viability were assessed using MTT assay, and AMPK phosphorylation, the expressions of NOX4, NRF2, SOD2, BAX, Bcl-2, and cleaved caspase-3 were detected with Western blotting. Male C57BL/6 mice were randomized into sham operation, I/R, I/R+colchicine, and I/R+colchicine+dorsomorphin (DSMP) groups. After the treatments, myocardial expressions of p-AMPK/AMPK, 8-OHdG, cleaved caspase-3, mitochondrial BAX (Mito-BAX), and cytoplasmic cytochrome C (Cyt-Cyto C) were examined and cardiac functions, infarct area, ATP content, and serum levels of lactic dehydrogenase (LDH) and cardiac troponin T (cTnT) levels were assessed. In H9C2 cells, H/R exposure significantly reduced AMPK phosphorylation and expressions of NRF2, SOD2, and Bcl-2, lowered cell viability, and up-regulated the expressions of NOX4, BAX, and cleaved caspase-3 (P < 0.05), and these changes were obviously alleviated by colchicine treatment (P < 0.05). In the mouse models, myocardial I/R injury significantly reduced myocardial AMPK phosphorylation level, ATP content, and expressions of NRF2, SOD2 and Bcl-2, caused cardiac function impairment, enhanced NOX4, Mito-BAX, Cyt-Cyto C, BAX, 8-OHdG, and cleaved caspase-3 expressions, and increased infarct area and serum LDH and cTnT levels (P < 0.05). Colchicine treatment significantly reversed the damaging effects of I/R (P < 0.05), but its protective effects was obviously antagonized by DSMP (P < 0.05). Colchicine alleviates myocardial I/R injury and protects cardiac function in mice by reducing myocardial oxidative stress and apoptosis via activating AMPK.

Anthocyanins and their metabolites promote white adipose tissue beiging by regulating mitochondria thermogenesis and dynamics

High-fat diet (HFD) consumption and excess nutrient availability can cause alterations in mitochondrial function and dynamics. We previously showed that anthocyanins (AC) decreased HFD-induced body weight gain and fat deposition. This study investigated: i) the capacity of AC to mitigate HFD-induced alterations in mitochondrial dynamics, biogenesis, and thermogenesis in mouse subcutaneous white adipose tissue (sWAT), and ii) the underlying mechanisms of action of cyanidin-3-O-glucoside (C3G), delphinidin-3-O-glucoside (D3G), and their gut metabolites on mitochondria function/dynamics in 3T3-L1 adipocytes treated with palmitate. Mice were fed control or HFD diets, added or not with 40 mg AC/kg body weight (BW). Compared to control and AC-supplemented mice, HFD-fed mice had fewer sWAT mitochondria that presented alterations of their architecture. AC supplementation prevented HFD-induced decrease of proteins involved in mitochondria biogenesis (PPARγ, PRDM16 and PGC-1α), and thermogenesis (UCP-1), and decreased AMPK phosphorylation. AC supplementation also restored the alterations in sWAT mitochondrial dynamics (Drp-1, OPA1, MNF-2, and Fis-1) and mitophagy (BNIP3L/NIX) caused by HFD consumption. In mature 3T3-L1, C3G, D3G, and their metabolites protocatechuic acid (PCA), 4-hydroxybenzaldehyde (HB), and gallic acid (GA) differentially affected palmitate-mediated decreased cAMP, PKA, AMPK, and SIRT-1 signaling pathways. C3G, D3G, and metabolites also prevented palmitate-mediated decreased expression of PPARγ, PRDM16, PGC-1α, and UCP1. Results suggest that consumption of select AC, i.e. cyanidin and delphinidin, could promote sWAT mitochondriogenesis and improve mitochondria dynamics in the context of HFD/obesity-induced dysmetabolism in part by regulating PKA, AMPK, and SIRT-1 signaling pathways.