Sex-specific role of sensory neuron LKB1 on metabolic stress-induced mechanical hypersensitivity and mitochondrial respiration.

Pain disorders induce metabolic stress in peripheral sensory neurons by reducing mitochondrial output, shifting cellular metabolism, and altering energy use. These processes implicate neuronal metabolism as an avenue for creating novel therapeutics. Liver kinase B1 (LKB1) mediates the cellular response to metabolic stress by inducing the 5'-adenosine monophosphate activated kinase (AMPK) pathway. The LKB1-AMPK pathway increases energy-producing processes, including mitochondrial output. These processes inhibit pain by directly or indirectly restoring energetic balance within a cell. Although the LKB1-AMPK pathway has been linked to pain relief, it is not yet known which cell is responsible for this property, as well any direct ties to cellular metabolism. To elucidate this, we developed a genetic mouse model where LKB1 is selectively removed from Nav1.8+ pain sensory neurons and metabolically stressed them by fasting for 24 h. We found females, but not males, had neuron-specific, LKB1-dependent restoration of metabolic stress-induced mitochondrial metabolism. This was reflected in mechanical hypersensitivity, where the absence of LKB1 led to hypersensitivity in female, but not male, animals. This discrepancy suggests a sex- and cell-specific contribution to LKB1-dependent fasting-induced mechanical hypersensitivity. Although our data represent a potential role for LKB1 in anti-pain pathways in a metabolic-specific manner, more must be done to investigate these sex differences.

Oral administration of berberine limits post-traumatic osteoarthritis development and associated pain via AMP-activated protein kinase (AMPK) in mice.

OBJECTIVE: We investigated the effect of berberine, a natural plant product that can activate AMP-activated protein kinase (AMPK), on Osteoarthritis (OA) development and associated pain in mice. DESIGN: Human primary knee chondrocytes were utilized to investigate how AMPK is activated by berberine. Both global knockout (KO) of AMPKα1 and congenic wild type (WT) mice were subjected to the post-traumatic OA through destabilization of medial meniscus (DMM) surgery. Two weeks after surgery, the mice were randomly divided into two groups with one group receiving berberine chloride daily via drinking water and were sacrificed at 6 and 12 weeks after surgery. OA severity was assessed by histological and histomorphometric analyses of cartilage degradation, synovitis, and osteophyte formation. OA-associated pain behavior was also determined. Immunohistochemistry (IHC) analyses were carried out to examine changes in AMPK signaling. RESULTS: Berberine induced phosphorylation of AMPKα (Thr172) via liver kinase B1 (LKB1), the major upstream kinase of AMPK, in chondrocytes in vitro. Both WT and AMPKα1KO developed OA and associated pain post DMM surgery. However, treatment with berberine significantly reduced severity of OA and associated pain in WT but not AMPKα1KO mice. IHC analysis of WT DMM knee cartilage further revealed that berberine inhibited concomitant loss of expression and phosphorylation of AMPKα and expression of SIRT1 and SIRT3, suggesting an important role of activation of AMPK signaling in mediating beneficial effect of berberine. CONCLUSIONS: Berberine acts through AMPK to reduce joint structural damage and pain associated with post-traumatic OA in mice in vivo.

Small activating RNA-activated NIS gene promotes 131I uptake and inhibits thyroid cancer via AMPK/mTOR pathway.

BACKGROUND: Sodium/iodide symporter (NIS) acts as a vital role in regulation of iodide uptake in thyroid cancer. However, the efficient approach to increase NIS expression and the mechanism of NIS-mediated iodide uptake in thyroid cancer remain unclear. METHODS: Small activating RNA (saRNA) was used to promote NIS expression. And the cell viability, apoptosis, and autophagy were detected using Cell count-kit 8 (CCK-8), Annexin V-FITC/PI double staining, and GFP-LC3 immunofluorescence assays, respectively. The protein levels of caspase 3, Bax, Bcl-2, ATG5, ATG12, LC3B Ⅱ to LC3B Ⅰ, Beclin 1, P62, AMPK, mTOR, P70S6K, actin, and phosphorylation of AMPK, mTOR, P70S6K were determined by western blotting. Moreover, a nude murine node with transplanted NC-dsRNA or NIS-482-transfected SW579 cells was used to examine the effect of NIS-mediated autophagy in vivo. And the levels of caspase 3 and ki67 were examined by immunohistochemical staining assay. RESULTS: saRNA mediated NIS mRNA and protein upregulated in SW579 cells. saRNA-mediated NIS expression inhibited cell proliferation, induced apoptosis and autophagy, and promoted iodide uptake in SW579 cells. Moreover, the effects of NIS on cells were enhanced by autophagy activator Rapamycin whereas reversed by autophagy inhibitor 3-Methyladenine (3-MA). For mechanism analysis, we found that NIS upregulation exerted the effects on cell proliferation, apoptosis, autophagy, and iodide uptake via regulating AMPK/mTOR pathway. We also demonstrated that saRNA-mediated NIS expression promoted iodide uptake in vivo. CONCLUSION: saRNA-mediated NIS expression acted as a critical role in increasing iodide uptake via AMPK/mTOR pathway in thyroid cancer.

LKB1 deficiency upregulates RELM-α to drive airway goblet cell metaplasia.

Targeting airway goblet cell metaplasia is a novel strategy that can potentially reduce the chronic obstructive pulmonary disease (COPD) symptoms. Tumor suppressor liver kinase B1 (LKB1) is an important regulator of the proliferation and differentiation of stem/progenitor cells. In this study, we report that LKB1 expression was downregulated in the lungs of patients with COPD and in those of cigarette smoke-exposed mice. Nkx2.1Cre; Lkb1f/f mice with conditional loss of Lkb1 in mouse lung epithelium displayed airway mucus hypersecretion and pulmonary macrophage infiltration. Single-cell transcriptomic analysis of the lung tissues from Nkx2.1Cre; Lkb1f/f mice further revealed that airway goblet cell differentiation was altered in the absence of LKB1. An organoid culture study demonstrated that Lkb1 deficiency in mouse airway (club) progenitor cells promoted the expression of FIZZ1/RELM-α, which drove airway goblet cell differentiation and pulmonary macrophage recruitment. Additionally, monocyte-derived macrophages in the lungs of Nkx2.1Cre; Lkb1f/f mice exhibited an alternatively activated M2 phenotype, while expressing RELM-α, which subsequently aggravated airway goblet cell metaplasia. Our findings suggest that the LKB1-mediated crosstalk between airway progenitor cells and macrophages regulates airway goblet cell metaplasia. Moreover, our data suggest that LKB1 agonists might serve as a potential therapeutic option to treat respiratory disorders associated with goblet cell metaplasia.

Sanse Powder Essential Oil Nanoemulsion Negatively Regulates TRPA1 by AMPK/mTOR Signaling in Synovitis: Knee Osteoarthritis Rat Model and Fibroblast-Like Synoviocyte Isolates.

Synovitis is the primary driving factor for the occurrence and development of knee osteoarthritis (KOA) and fibroblast-like synoviocytes (FLSs) and plays a crucial role during this process. Our previous works revealed that transient receptor potential ankyrin 1 (TRPA1) ion channels mediate the amplification of KOA synovitis. In recent years, essential oils have been proved to have blocking effect on transient receptor potential channels. Meanwhile, the therapeutic effect of Sanse Powder on KOA synovitis has been confirmed in clinical trials and basic studies; although, the mechanism remains unclear. In the present study, Sanse Powder essential oil nanoemulsion (SP-NEs) was prepared, and then chemical composition, physicochemical properties, and stability were investigated. Besides, both in MIA-induced KOA rats and in LPS-stimulated FLSs, we investigated whether SP-NES could alleviate KOA synovitis by interfering with AMP-activated protein kinase- (AMPK-) mammalian target of rapamycin (mTOR), an energy sensing pathway proved to negatively regulate the TRPA1. Our research shows that the top three substances in SP-NEs were tumerone, delta-cadinene, and Ar-tumerone, which accounted for 51.62% of the total, and should be considered as the main pharmacodynamic ingredient. Less inflammatory cell infiltration and type I collagen deposition were found in the synovial tissue of KOA rats treated with SP-NEs, as well as the downregulated expressions of interleukin (IL)-1ß, IL-18, and TRPA1. Besides, SP-NEs increased the phosphorylation level of AMPK and decreased the phosphorylation level of mTOR in the KOA model, and SP-NEs also upregulated expressions of peroxisome proliferator-activated receptor-gamma (PPARγ) and PPARγ coactivator-1α and downstream signaling molecules of AMPK-mTOR in vivo and in vitro. To conclude, a kind of Chinese herbal medicine for external use which is effective in treating synovitis of KOA was extracted and prepared into essential oil nanoemulsion with stable properties in the present study. It may alleviate synovitis in experimental KOA through the negative regulation of TRPA1 by AMPK-mTOR signaling.

Folliculin impairs breast tumor growth by repressing TFE3-dependent induction of the Warburg effect and angiogenesis.

Growing tumors exist in metabolically compromised environments that require activation of multiple pathways to scavenge nutrients to support accelerated rates of growth. The folliculin (FLCN) tumor suppressor complex (FLCN, FNIP1, FNIP2) is implicated in the regulation of energy homeostasis via 2 metabolic master kinases: AMPK and mTORC1. Loss-of-function mutations of the FLCN tumor suppressor complex have only been reported in renal tumors in patients with the rare Birt-Hogg-Dube syndrome. Here, we revealed that FLCN, FNIP1, and FNIP2 are downregulated in many human cancers, including poor-prognosis invasive basal-like breast carcinomas where AMPK and TFE3 targets are activated compared with the luminal, less aggressive subtypes. FLCN loss in luminal breast cancer promoted tumor growth through TFE3 activation and subsequent induction of several pathways, including autophagy, lysosomal biogenesis, aerobic glycolysis, and angiogenesis. Strikingly, induction of aerobic glycolysis and angiogenesis in FLCN-deficient cells was dictated by the activation of the PGC-1α/HIF-1α pathway, which we showed to be TFE3 dependent, directly linking TFE3 to Warburg metabolic reprogramming and angiogenesis. Conversely, FLCN overexpression in invasive basal-like breast cancer models attenuated TFE3 nuclear localization, TFE3-dependent transcriptional activity, and tumor growth. These findings support a general role of a deregulated FLCN/TFE3 tumor suppressor pathway in human cancers.

Calcium/Calmodulin Dependent Protein Kinase Kinase 2 Regulates the Expansion of Tumor-Induced Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSCs) are a hetero geneous group of cells, which can suppress the immune response, promote tumor progression and impair the efficacy of immunotherapies. Consequently, the pharmacological targeting of MDSC is emerging as a new immunotherapeutic strategy to stimulate the natural anti-tumor immune response and potentiate the efficacy of immunotherapies. Herein, we leveraged genetically modified models and a small molecule inhibitor to validate Calcium-Calmodulin Kinase Kinase 2 (CaMKK2) as a druggable target to control MDSC accumulation in tumor-bearing mice. The results indicated that deletion of CaMKK2 in the host attenuated the growth of engrafted tumor cells, and this phenomenon was associated with increased antitumor T cell response and decreased accumulation of MDSC. The adoptive transfer of MDSC was sufficient to restore the ability of the tumor to grow in Camkk2-/- mice, confirming the key role of MDSC in the mechanism of tumor rejection. In vitro studies indicated that blocking of CaMKK2 is sufficient to impair the yield of MDSC. Surprisingly, MDSC generated from Camkk2-/- bone marrow cells also showed a higher ability to terminally differentiate toward more immunogenic cell types (e.g inflammatory macrophages and dendritic cells) compared to wild type (WT). Higher intracellular levels of reactive oxygen species (ROS) accumulated in Camkk2-/- MDSC, increasing their susceptibility to apoptosis and promoting their terminal differentiation toward more mature myeloid cells. Mechanistic studies indicated that AMP-activated protein kinase (AMPK), which is a known CaMKK2 proximal target controlling the oxidative stress response, fine-tunes ROS accumulation in MDSC. Accordingly, failure to activate the CaMKK2-AMPK axis can account for the elevated ROS levels in Camkk2-/- MDSC. These results highlight CaMKK2 as an important regulator of the MDSC lifecycle, identifying this kinase as a new druggable target to restrain MDSC expansion and enhance the efficacy of anti-tumor immunotherapy.

Inositol serves as a natural inhibitor of mitochondrial fission by directly targeting AMPK.

Mitochondrial dynamics regulated by mitochondrial fusion and fission maintain mitochondrial functions, whose alterations underline various human diseases. Here, we show that inositol is a critical metabolite directly restricting AMPK-dependent mitochondrial fission independently of its classical mode as a precursor for phosphoinositide generation. Inositol decline by IMPA1/2 deficiency elicits AMPK activation and mitochondrial fission without affecting ATP level, whereas inositol accumulation prevents AMPK-dependent mitochondrial fission. Metabolic stress or mitochondrial damage causes inositol decline in cells and mice to elicit AMPK-dependent mitochondrial fission. Inositol directly binds to AMPKγ and competes with AMP for AMPKγ binding, leading to restriction of AMPK activation and mitochondrial fission. Our study suggests that the AMP/inositol ratio is a critical determinant for AMPK activation and establishes a model in which AMPK activation requires inositol decline to release AMPKγ for AMP binding. Hence, AMPK is an inositol sensor, whose inactivation by inositol serves as a mechanism to restrict mitochondrial fission.

AMPK Phosphorylation Is Controlled by Glucose Transport Rate in a PKA-Independent Manner.

To achieve growth, microbial organisms must cope with stresses and adapt to the environment, exploiting the available nutrients with the highest efficiency. In Saccharomyces cerevisiae, Ras/PKA and Snf1/AMPK pathways regulate cellular metabolism according to the supply of glucose, alternatively supporting fermentation or mitochondrial respiration. Many reports have highlighted crosstalk between these two pathways, even without providing a comprehensive mechanism of regulation. Here, we show that glucose-dependent inactivation of Snf1/AMPK is independent from the Ras/PKA pathway. Decoupling glucose uptake rate from glucose concentration, we highlight a strong coordination between glycolytic metabolism and Snf1/AMPK, with an inverse correlation between Snf1/AMPK phosphorylation state and glucose uptake rate, regardless of glucose concentration in the medium. Despite fructose-1,6-bisphosphate (F1,6BP) being proposed as a glycolytic flux sensor, we demonstrate that glucose-6-phosphate (G6P), and not F1,6BP, is involved in the control of Snf1/AMPK phosphorylation state. Altogether, this study supports a model by which Snf1/AMPK senses glucose flux independently from PKA activity, and thanks to conversion of glucose into G6P.

Impairment of AMPK-α2 augments detrusor contractions in bladder ischemia.

PURPOSE: Ischemia disrupts cellular energy homeostasis. Adenosine monophosphate-activated protein kinase alpha-2 (AMPK-α2) is a subunit of AMPK that senses cellular energy deprivation and signals metabolic stress. Our goal was to examine the expression levels and functional role of AMPK-α2 in bladder ischemia. MATERIALS AND METHODS: Iliac artery atherosclerosis and bladder ischemia were engendered in apolipoprotein E knockout rats by partial arterial endothelial denudation using a balloon catheter. After eight weeks, total and phosphorylated AMPK-α2 expression was analyzed by western blotting. Structural integrity of AMPK-α2 protein was assessed by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). Functional role of AMPK-α2 was examined by treating animals with the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D ribofuranoside (AICAR). Tissue contractility was measured in the organ bath and bladder nerve density was examined by immunostaining. RESULTS: Total AMPK-α2 expression increased in bladder ischemia, while phosphorylated AMPK-α2 was significantly downregulated. LC-MS/MS suggested post-translational modification of AMPK-α2 functional domains including phosphorylation sites, suggesting accumulation of catalytically inactive AMPK-α2 in bladder ischemia. Treatment of rats with AICAR diminished the force of overactive detrusor contractions and increased bladder capacity but did not have a significant effect on the frequency of bladder contractions. AICAR diminished contractile reactivity of ischemic tissues in the organ bath and prevented loss of nerve fibers in bladder ischemia. CONCLUSIONS: Ischemia induces post-translational modification of AMPK-α2 protein. Impairment of AMPK-α2 may contribute to overactive detrusor contractions and loss of nerve fibers in bladder ischemia. AMPK activators may have therapeutic potential against detrusor overactivity and neurodegeneration in bladder conditions involving ischemia.

Pectic Oligogalacturonide Facilitates the Synthesis and Activation of Adiponectin to Improve Hepatic Lipid Oxidation.

SCOPE: Adiponectin (ADPN), a kind of adipokines, plays an important role in the regulation of lipid metabolism. The objective of this study is focused on the ADPN to investigate the functional mechanisms of pectin oligosaccharide (POS) from hawthorn fruit in the improvement of hepatic fatty acid oxidation. METHOD AND RESULTS: High-fat fed mice are used in this experiment. POS is administrated with the doses of 0.25, 0.75, and 1.5 g kg-1 diet, respectively. The results demonstrate that gene and protein expressions of ADPN synthesis regulators involved in PKA/ERK/CREB and C/EBPα/PPARγ pathways are upregulated by POS administration. POS also activates the AdiopR1/AMPKα/PGC1 and AdipoR2/PPARα signaling pathways to improve the fatty acid oxidation in the liver, which is further accelerated by the enhancement of mitochondrial functions. CONCLUSION: POS can act as an ADPN activator to improve lipid metabolism, leading it to the applications of biomedical and functional foods for ameliorating chronic liver diseases resulted from a high-energy diet.

Modeling High-Grade Serous Ovarian Carcinoma Using a Combination of In Vivo Fallopian Tube Electroporation and CRISPR-Cas9-Mediated Genome Editing.

Ovarian cancer is the most lethal gynecologic cancer to date. High-grade serous ovarian carcinoma (HGSOC) accounts for most ovarian cancer cases, and it is most frequently diagnosed at advanced stages. Here, we developed a novel strategy to generate somatic ovarian cancer mouse models using a combination of in vivo electroporation and CRISPR-Cas9-mediated genome editing. Mutation of tumor suppressor genes associated with HGSOC in two different combinations (Brca1, Tp53, Pten with and without Lkb1) resulted in successfully generation of HGSOC, albeit with different latencies and pathophysiology. Implementing Cre lineage tracing in this system enabled visualization of peritoneal micrometastases in an immune-competent environment. In addition, these models displayed copy number alterations and phenotypes similar to human HGSOC. Because this strategy is flexible in selecting mutation combinations and targeting areas, it could prove highly useful for generating mouse models to advance the understanding and treatment of ovarian cancer. SIGNIFICANCE: This study unveils a new strategy to generate genetic mouse models of ovarian cancer with high flexibility in selecting mutation combinations and targeting areas.

Adenylate Kinase 4 Promotes Inflammatory Gene Expression via Hif1α and AMPK in Macrophages.

Macrophages comprise the front line of defense against various pathogens. Classically activated macrophages (M1), induced by IFN-γ and LPS, highly express inflammatory cytokines and contribute to inflammatory processes. By contrast, alternatively activated macrophages (M2) are induced by IL-4 and IL-13, produce IL-10, and display anti-inflammatory activity. Adenylate kinase 4 (Ak4), an enzyme that transfers phosphate group among ATP/GTP, AMP, and ADP, is a key modulator of ATP and maintains the homeostasis of cellular nucleotides which is essential for cell functions. However, its role in regulating the function of macrophages is not fully understood. Here we report that Ak4 expression is induced in M1 but not M2 macrophages. Suppressing the expression of Ak4 in M1 macrophages with shRNA or siRNA enhances ATP production and decreases ROS production, bactericidal ability and glycolysis in M1 cells. Moreover, Ak4 regulates the expression of inflammation genes, including Il1b, Il6, Tnfa, Nos2, Nox2, and Hif1a, in M1 macrophages. We further demonstrate that Ak4 inhibits the activation of AMPK and forms a positive feedback loop with Hif1α to promote the expression of inflammation-related genes in M1 cells. Furthermore, RNA-seq analysis demonstrates that Ak4 also regulates other biological processes in addition to the expression of inflammation-related genes in M1 cells. Interestingly, Ak4 does not regulate M1/M2 polarization. Taken together, our study uncovers a potential mechanism linking energy consumption and inflammation in macrophages.

Extracellular Superoxide Dismutase Prevents Skin Aging by Promoting Collagen Production through the Activation of AMPK and Nrf2/HO-1 Cascades.

With aging, the skin becomes thin and drastically loses collagen. Extracellular superoxide dismutase (EC-SOD), also known as superoxide dismutase (SOD) 3, is the major SOD in the extracellular matrix of the tissues and is well-known to maintain the reduction‒oxidation homeostasis and matrix components of such tissues. However, the role of EC-SOD in aging-associated reductions of skin thickness and collagen production is not well-studied. In this study, we compared the histological differences in the dorsal skin of EC-SOD‒overexpressing transgenic mice (Sod3+/+) of different age groups with that in wild-type mice and also determined the underlying signaling mechanism. Our data showed that the skin thickness in Sod3+/+ mice significantly increased with aging compared with that in wild-type male mice. Furthermore, Sod3+/+ mice had promoted collagen production through the activation of adenosine monophosphate-activated protein kinase and Nrf2/HO-1 pathways in aged mice. Interestingly, subcutaneous injection of adeno-associated virus‒overexpressing EC-SOD exhibited increased skin thickness and collagen expression. Furthermore, combined recombinant EC-SOD and dihydrotestosterone treatment synergistically elevated collagen production through the activation of TGFß in human dermal fibroblasts. Altogether, these results showed that EC-SOD prevents skin aging by promoting collagen production in vivo and in vitro. Therefore, we propose that EC-SOD may be a potential therapeutic target for antiaging in the skin.

α-Amyrin induces GLUT4 translocation mediated by AMPK and PPARδ/γ in C2C12 myoblasts.

α-Amyrin, a natural pentacyclic triterpene, has an antihyperglycemic effect in mice and dual PPARδ/γ action in 3T3-L1 adipocytes, and potential in the control of type 2 diabetes (T2D). About 80% of glucose uptake occurs in skeletal muscle cells, playing a significant role in insulin resistance (IR) and T2D. Peroxisome-proliferator activated receptors (PPARs), in particular PPARδ and PPARγ, are involved in the regulation of lipids and carbohydrates and, along with adenosine-monophosphate (AMP) - activated protein kinase (AMPK) and protein kinase B (Akt), are implicated in translocation of glucose transporter 4 (GLUT4); however, it is still unknown whether α-amyrin can affect these pathways in skeletal muscle cells. Our objective was to determine the action of α-amyrin in PPARδ, PPARγ, AMPK, and Akt in C2C12 myoblasts. The expression of PPARδ, PPARγ, fatty acid transporter protein (FATP), and GLUT4 was quantified using reverse transcription quantitative PCR and Western blot. α-Amyrin increased these markers along with phospho-AMPK (p-AMPK) but not p-Akt. Molecular docking showed that α-amyrin acts as an AMPK-allosteric activator, and may be related to GLUT4 translocation, as evidenced by confocal microscopy. These data support that α-amyrin could have an insulin-mimetic action in C2C12 myoblasts and should be considered as a bioactive molecule for new multitarget drugs with utility in T2D and other metabolic diseases.

Metformin attenuates ischemia/reperfusion-induced apoptosis of cardiac cells by downregulation of p53/microRNA-34a via activation of SIRT1.

Metformin has been demonstrated to be beneficial for the treatment of an impaired myocardium as a result of ischemia/reperfusion (I/R) injury, and miR-34a may be involved in this process. The aim of the present study was to determine the mechanisms by which metformin attenuated myocardial I/R injury-induced apoptosis. In the in vivo I/R model using Sprague-Dawley rats, metformin reduced the area of damaged myocardium and serum creatine MB isoform (CKMB) activity resulting in protection of the myocardium. Metformin also reduced apoptosis and the expression of apoptosis associated proteins, including caspase 3 and cleaved caspase, and decreased the expression of miR-34a, which is upregulated during I/R injury, which in turn resulted in corresponding changes in expression of Bcl-2, a direct target of miR-34a both in vitro and in vivo. To further examine the role of miR-34a in this process, H9C2 cells were transfected by a miR-34a mimic and inhibitor. Overexpression of miR-34a increased apoptosis in H9C2 cells induced by oxygen-glucose deprivation/recovery and knockdown of miR-34a expression-reduced apoptosis under the same conditions. Therefore, the effect of metformin on miR-34a in vitro were assessed. Metformin decreased the deacetylation activity of silent information regulator 1 resulting in reduced Ac-p53 levels, which reduced the levels of pri-miR-34a, and thus in turn reduced miR-34a levels. To confirm these results clinically, 90 patients with ST-segment elevation myocardial infarction following percutaneous coronary intervention were recruited. Patients who took metformin regularly before infarction had lower miR-34a levels and lower serum CKMB activity. Metformin also improved the sum ST-segment recovery following I/R injury. In conclusion, metformin may be helpful in the treatment of myocardial I/R.

Ghrelin protects against rotenone-induced cytotoxicity: Involvement of mitophagy and the AMPK/SIRT1/PGC1α pathway.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, characterized by the loss of dopaminergic neurons in the substantia nigra and the deposition of Lewy bodies. Mitochondrial dysfunction, oxidative stress, and autophagy dysfunction are involved in the pathogenesis of PD. Ghrelin is a brain-gut peptide that has been reported that protected against 1-methyl-4-phenyl-1,2,3,6- tetrahydropyran (MPTP)/MPP+-induced toxic effects. In the present work, human neuroblastoma SH-SY5Y cells were exposed to rotenone as a PD model to explore the underlying mechanism of ghrelin. We found that ghrelin inhibited rotenone-induced cytotoxicity, mitochondrial dysfunction, and apoptosis by improving cell viability, increasing the ratio of red/green of JC-1, inhibiting the production of reactive oxidative species (ROS), and regulating Bcl-2, Bax, Cytochrome c, caspase-9, and caspase-3 expression. Besides, ghrelin promoted mitophagy accompanied by up-regulating microtubule-associated protein 1 Light Chain 3B-II/I(LC3B-II/I) and Beclin1 but decreasing the expression of p62. Moreover, ghrelin promoted PINK1/Parkin mitochondrial translocation. Additionally, we investigated that ghrelin activated the AMPK/SIRT1/PGC1α pathway and pharmacological inhibition of AMPK and SIRT1 abolished the cytoprotection of ghrelin, decreased the level of mitophagy, and PINK1/Parkin mitochondrial translocation. Taken together, our findings suggested that mitophagy and AMPK/SIRT1/PGC1α pathways were related to the cytoprotection of ghrelin. These findings provided novel insights into the underlying mechanisms of ghrelin, further mechanistic studies on preclinical and clinical levels are required to be conducted with ghrelin to avail and foresee it as a potential agent in the treatment and management of PD.

Blockage of AMPK-ULK1 pathway mediated autophagy promotes cell apoptosis to increase doxorubicin sensitivity in breast cancer (BC) cells: an in vitro study.

BACKGROUND: Activation of autophagy flux contributed to resistance of breast cancer (BC) cells to current chemotherapeutic drugs, which seriously limited their therapeutic efficacy and facilitated BC recurrence in clinic. However, the detailed mechanisms are still not fully understood. In the present study, we identified that inactivation of AMPK-ULK1 signaling cascade mediated protective autophagy sensitized BC cells to doxorubicin in vitro. METHODS: Cell counting kit-8 (CCK-8) assay and colony formation assay were performed to evaluate cell proliferation abilities. Trypan blue staining assay was used to examine cell viability, and Annexin V-FITC/PI double staining method was conducted to determine cell apoptosis. The autophagosomes in BC cells were observed and photographed by electronic microscope (EM). Western Blot analysis was employed to examine genes expressions at protein levels. RESULTS: The parental doxorubicin-sensitive BC (DS-BC) cells were exposed to increasing concentrations of doxorubicin to establish doxorubicin-resistant BC (DR-BC) cells, and the DR-BC cells were much more resistant to high-dose doxorubicin treatment compared to the DS-BC cells. Interestingly, high-dose doxorubicin specifically increased LC3B-II/I ratio, promoted autophagosomes formation and decreased p62 expression levels to facilitate autophagy in DR-BC cells, instead of DS-BC cells, and the autophagy inhibitor 3-methyladenine (3-MA) enhanced the cytotoxic effects of high-dose doxorubicin on DR-BC cells. In addition, we proved that high-dose doxorubicin triggered protective autophagy in DR-BC cells by activating AMPK-ULK1 pathway. Functionally, high-dose doxorubicin increased the expression levels of phosphorylated AMPK (p-AMPK) and ULK1 (p-ULK1) to activate AMPK-ULK1 pathway in DR-BC cells, and the inhibitors for AMPK (compound C) and ULK1 (SBI-0206965) blocked autophagy to promote cell death and slow down cell growth in DR-BC cells treated with high-dose doxorubicin. CONCLUSIONS: Collectively, our in vitro data indicated that blockage of AMPK-ULK1 signaling cascade mediated protective autophagy might be a promising strategy to increase doxorubicin sensitivity for BC treatment.

Mesenchymal stem cells promote chemoresistance by activating autophagy in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is a type of cancer that is difficult to cure; chemoresistance of cholangiocarcinoma cells affect the prognosis of patients who cannot be treated with surgery. The mechanism underlying this chemoresistance remains unknown. Mesenchymal stem cells (MSCs) are known to be important components of the tumor microenvironment. In the present study, a large number of MSCs were observed to infiltrate the tumor sites of ICC; thus, MSCs were isolated from ICC tumor tissues. It was revealed that herpesvirus entry mediator (HVEM) was overexpressed in ICC­MSCs. The present study then investigated the role of HVEM­overexpressing MSCs in the chemoresistance of cholangiocarcinoma cells. It was demonstrated that HVEM­overexpressing MSCs could support cell survival of chemotherapeutic cholangiocarcinoma cells and inhibited their apoptosis. Further investigations revealed that HVEM­overexpressing MSCs could secrete IL­6 and also activated AMPK/mTOR­dependent autophagy of cholangiocarcinoma cells. Thus, it was concluded that ICC­MSC­induced autophagy is the primary cause of chemoresistance in ICC.

Long-term T cell fitness and proliferation is driven by AMPK-dependent regulation of reactive oxygen species.

The AMP-activated kinase (AMPK) is a major energy sensor metabolic enzyme that is activated early during T cell immune responses but its role in the generation of effector T cells is still controversial. Using both in vitro and in vivo models of T cell proliferation, we show herein that AMPK is dispensable for early TCR signaling and short-term proliferation but required for sustained long-term T cell proliferation and effector/memory T cell survival. In particular, AMPK promoted accumulation of effector/memory T cells in competitive homeostatic proliferation settings. Transplantation of AMPK-deficient hematopoïetic cells into allogeneic host recipients led to a reduced graft-versus-host disease, further bolstering a role for AMPK in the expansion and pathogenicity of effector T cells. Mechanistically, AMPK expression enhances the mitochondrial membrane potential of T cells, limits reactive oxygen species (ROS) production, and resolves ROS-mediated toxicity. Moreover, dampening ROS production alleviates the proliferative defect of AMPK-deficient T cells, therefore indicating a role for an AMPK-mediated ROS control of T cell fitness.