An AMPK agonist suppresses the progress of colorectal cancer by regulating the polarization of TAM to M1 through inhibition of HIF-1α and mTOR signal pathway.

OBJECTIVE: This study aimed to evaluate the impact of an adenosine monophosphate-activated protein kinase (AMPK) agonist, metformin (MET), on the antitumor effects of macrophages and to determine the underlying mechanism involved in the process. MATERIALS AND METHODS: M0 macrophages were derived from phorbol-12-myristate-13-acetate-stimulated THP-1 cells. RESULTS: The levels of tumor necrosis factor-alpha (TNF-α) and human leukocyte antigen-DR (HLA-DR) were decreased in macrophages incubated with HCT116 cells, whereas those of arginase-1 (Arg-1), CD163, and CD206 were elevated; these effects were reversed by MET. The transfection of small interfering (si) RNA abrogated the influence of MET on the expression of the M1/M2 macrophage biomarkers. MET significantly suppressed the proliferation and migration abilities of HCT116 cells incubated with M0 macrophages; these actions were reversed by siRNA transfection against AMPK. The hypoxia-inducible factor 1-alpha (HIF-1α), phosphorylated protein kinase B (p-AKT), and phosphorylated mammalian target of rapamycin (p-mTOR) levels were reduced by the introduction of MET and promoted by siRNA transfection against AMPK. In addition, the levels of HIF-1α, p-AKT, and p-mTOR suppressed by MET were markedly increased following the transfection of siRNA against AMPK. CONCLUSION: These findings indicate that MET can repress the progression of colorectal cancer by transforming tumor-associated macrophages to the M1phenotype via inhibition of the HIF-1α and mTOR signaling pathways.

Identification of novel benzimidazole derivatives as highly potent AMPK activators with anti-diabetic profiles.

Diabetes is a global healthcare problem that affects more than 400 million people worldwide. Treatment for type 1 and 2 diabetes is expected by targeting adenosine monophosphate activated protein kinase, AMPK, a well-known master regulator of glucose. Many pharmaceutical companies have tried to identify AMPK activators but few direct AMPK activators with high potency for the ß2-AMPK isoform, which is important for glucose homeostasis, have been found. In addition, their chemical structure is limited to benzimidazole or indole derivatives bearing an aromatic substituent at the C5 position of the core structure. We describe herein our efforts to identify novel benzimidazole derivatives that directly activate the ß2-AMPK isoform. Our newly designed activator 14d bearing a 1-amino indanyl moiety at the C5 position of the core exhibited high in vitro potency and good pharmacokinetic profiles. A single oral dosing of 14d showed dose-dependent activation of AMPK and blood-glucose-lowering effects was observed in a diabetic animal model. In addition, chronic AMPK activation with 14d led to dose-dependent reduction in HbA1c of the animal model.

Differential Effects of Small Molecule Inhibitors on the Intracellular Chlamydia Infection.

Chlamydia are obligate intracellular bacteria that reside within a membrane-bound compartment called the chlamydial inclusion inside a eukaryotic host cell. These pathogens have a complex biphasic developmental cycle, which involves conversion between a replicating, but noninfectious, reticulate body (RB) and an infectious elementary body (EB). Small molecule inhibitors have been reported to have deleterious effects on the intracellular Chlamydia infection, but these studies have typically been limited in terms of assays and time points of analysis. We compared published and novel inhibitors and showed that they can differentially alter inclusion size, chlamydial number and infectious EB production, and that these effects can vary over the course of the intracellular infection. Our results provide the justification for analysis with multiple assays performed either at the end of the infection or over a time course. We also show that this approach has the potential to identify the particular step in the developmental cycle that is impacted by the inhibitor. We furthermore propose that the magnitude of inhibitor-induced progeny defects are best quantified and compared by using a new value called maximal progeny production (Progenymax). As a demonstration of the validity of this systematic approach, we applied it to inhibitors of Akt and AMPK, which are host kinases involved in lipid synthesis and cholesterol trafficking pathways. Both inhibitors reduced EB production, but Akt disruption primarily decreased RB-to-EB conversion while AMPK inhibition paradoxically enhanced RB replication. IMPORTANCE Chlamydia is the most reported cause of bacterial, sexually transmitted infection in the United States. This bacterium infects human cells and reproduces within a cytoplasmic inclusion via an unusual developmental cycle involving two specialized chlamydial forms. Small molecule compounds have been reported to negatively affect the inclusion as well as chlamydial replication and infectious progeny production, but we showed that these effects can be discordant and vary over the course of the 48- to 72-hour long intracellular infection. We propose approaches to analyze these nonuniform effects, including measurements at the end of the intracellular infection, and more detailed analysis with multiple assays performed over the course of the developmental cycle. We then applied this approach to investigate and compare the anti-chlamydial effects of two inhibitors that alter host lipid synthesis and cholesterol trafficking.

10-Gingerol, a natural AMPK agonist, suppresses neointimal hyperplasia and inhibits vascular smooth muscle cell proliferation.

Background: Abnormal proliferation of vascular smooth muscle cells (VSMCs) in the intimal region is a key event in the development of neointimal hyperplasia. 10-G, a bioactive compound found in ginger, exerted inhibitory effects on the proliferation of several cancer cells. However, the effect and mechanism of 10-G on neointimal hyperplasia are not clear. Purpose: To explore the suppressive effects of 10-G on the proliferation and migration of VSMCs, and investigate the underlying mechanisms. Methods: In vivo, a left common carotid artery ligation mouse model was used to observe the effects of neointimal formation through immunohistochemistry and hematoxylin-eosin staining. In vitro, the cell proliferation and migration of HASMCs and A7r5 cells were detected by MTS assay, EdU staining, wound healing assay, Transwell assay, and western blotting as well. Molecular docking, molecular dynamics simulations and surface plasmon resonance imaging were collectively used to evaluate the interaction of 10-G with AMP-activated protein kinase (AMPK). Compound C and si-AMPK were used to inhibit the expression of AMPK. Results: Treatment with 10-G significantly reduced neointimal hyperplasia in the left common carotid artery ligation mouse model. MST and EdU staining showed that 10-G inhibited the proliferation of VSMC cells A7r5 and HASMC. We also found that 10-G altered the expression of proliferation-related proteins, including CyclinD1, CyclinD2, CyclinD3, and CDK4. Molecular docking revealed that the binding energy between AMPK and 10-G is -7.4 kcal mol-1. Molecular simulations suggested that the binding between 10-G and AMPK is stable. Surface plasmon resonance imaging analysis also showed that 10-G has a strong binding affinity to AMPK (KD = 6.81 × 10-8 M). 10-G promoted AMPKα phosphorylation both in vivo and in vitro. Blocking AMPK by an siRNA or AMPK inhibitor pathway partly abolished the anti-proliferation effects of 10-G on VSMCs. Conclusion: These data showed that 10-G might inhibit neointimal hyperplasia and suppress VSMC proliferation by the activation of AMPK as a natural AMPK agonist.

Axonal Protection by Netarsudil, a ROCK Inhibitor, Is Linked to an AMPK-Autophagy Pathway in TNF-Induced Optic Nerve Degeneration.

Purpose: Netarsudil, a Rho kinase inhibitor with norepinephrine transport inhibitory effect, lowers intraocular pressure, however, its effect on axon damage remains to be elucidated. The aim of the current study was to investigate the effect of netarsudil on TNF-induced axon loss and to examine whether it affects phosphorylated-AMP-activated kinase (p-AMPK) and autophagy in the optic nerve. Methods: Intravitreal administration of TNF or TNF with netarsudil was carried out on rats and quantification of axon number was determined. Electron microscopy determined autophagosome numbers. Localization of p-AMPK expression was examined by immunohistochemistry. The changes in p62, LC3-II, and p-AMPK levels were estimated in the optic nerve by immunoblot analysis. The effect of an AMPK activator A769662 or an AMPK inhibitor dorsomorphin on axon number was evaluated. Results: Morphometric analysis revealed apparent protection by netarsudil against TNF-induced axon degeneration. Netarsudil increased autophagosome numbers inside axons. Netarsudil treatment significantly upregulated optic nerve LC3-II levels in both the TNF-treated eyes and the control eyes. Increased p62 protein level induced by TNF was significantly ameliorated by netarsudil. The netarsudil administration alone lessened p62 levels. Netarsudil significantly upregulated the optic nerve p-AMPK levels. A769662 exhibited obvious axonal protection against TNF-induced damage. A769662 treatment upregulated LC3-II levels and the increment of p62 level induced by TNF was significantly ameliorated by A769662. Immunohistochemical analysis revealed that p-AMPK is present in axons. Netarsudil-mediated axonal protection was significantly suppressed by dorsomorphin administration. Conclusions: Netarsudil upregulated p-AMPK and autophagy. Netarsudil-mediated axonal protection may be associated with upregulated p-AMPK.

Virus-mediated decrease of LKB1 activity in the mPFC diminishes stress-induced depressive-like behaviors in mice.

As a highly prevalent neuropsychiatric disorder worldwide, the pathophysiology of depression is not yet fully understood and based on multiple factors among which chronic stress is critical. Numerous previous studies have shown the role of central mammalian target of rapamycin complex 1 (mTORC1) signaling in depression. However, so far it remains elusive by which way chronic stress down-regulates the activity of central mTORC1. Liver kinase b1 (LKB1) has been demonstrated to regulate the activity of the mTORC1 signaling cascade by phosphorylating AMP activated protein kinase (AMPK). Here, this study aimed to explore whether LKB1 participates in depression by regulating the downstream AMPK-mTORC1 signaling, and various methods including mouse models of depression, western blotting and immunofluorescence were used together. Our results showed that chronic stress significantly enhanced the expression of both phosphorylated LKB1 and total LKB1 in the medial prefrontal cortex (mPFC) but not the hippocampus. Furthermore, genetic knockdown of LKB1 in the mPFC fully reversed not only the depressive-like behaviors induced by chronic stress in mice but also the effects of chronic stress on the activity of AMPK and the mTORC1 system. Taken together, this study preliminarily suggests that LKB1 in the mPFC could be a feasible target for antidepressants. This study also provides support for the potential use of LKB1 inhibition strategies against the chronic stress-related neuropsychiatric disorders.

Mangiferin Ameliorates HFD-Induced NAFLD through Regulation of the AMPK and NLRP3 Inflammasome Signal Pathways.

Nonalcoholic fatty liver disease (NAFLD) is closely related to glycolipid metabolism and liver inflammation. And there is no effective drug approved for its clinical therapy. In this study, we focused on mangiferin (Man) and explored its effects and mechanisms on NAFLD treatment based on the regulation of glycolipid metabolism and anti-inflammatory in vivo and in vitro. The results exhibited that Man can significantly attenuate liver injury, insulin resistance, and glucose tolerance in high-fat diet- (HFD-) induced NAFLD mice and significantly reduce fat accumulation and inflammation in hepatic tissue of NAFLD mice. The transcriptome level RNA-seq analysis showed that the significantly different expression genes between the Man treatment group and the HFD-induced NAFLD model group were mainly related to regulation of energy, metabolism, and inflammation in liver tissue. Furthermore, western blots, real-time PCR, and immunohistochemistry experiments confirmed that Man significantly activated the AMPK signal pathway and inhibited NLRP3 inflammasome activation and pyroptosis in NAFLD mice. In in vitro cell experiments, we further confirmed that Man can promote glucose consumption and reduce intracellular triglyceride (TG) accumulation induced by free fatty acids in HepG2 cells and further that it can be blocked by AMPK-specific inhibitors. Western blot results showed that Man upregulated p-AMPKα levels and exhibited a significant AMPK activation effect, which was blocked by compound C. At the same time, Man downregulated the expression of NLRP3 inflammasome-related proteins and inhibited the activation of NLRP3 inflammasome, alleviating cell pyroptosis and inflammation effects. These results indicate that Man anti-NAFLD activity is mediated through its regulation of glucolipid metabolism by AMPK activation and its anti-inflammatory effects by NLRP3 inflammasome inhibition. Our study indicates that Man is a promising prodrug for the therapy of NAFLD patients.

Serum- and glucocorticoid-induced kinase drives hepatic insulin resistance by directly inhibiting AMP-activated protein kinase.

A hallmark of type 2 diabetes (T2D) is hepatic resistance to insulin's glucose-lowering effects. The serum- and glucocorticoid-regulated family of protein kinases (SGK) is activated downstream of mechanistic target of rapamycin complex 2 (mTORC2) in response to insulin in parallel to AKT. Surprisingly, despite an identical substrate recognition motif to AKT, which drives insulin sensitivity, pathological accumulation of SGK1 drives insulin resistance. Liver-specific Sgk1-knockout (Sgk1Lko) mice display improved glucose tolerance and insulin sensitivity and are protected from hepatic steatosis when fed a high-fat diet. Sgk1 promotes insulin resistance by inactivating AMP-activated protein kinase (AMPK) via phosphorylation on inhibitory site AMPKαSer485/491. We demonstrate that SGK1 is dominant among SGK family kinases in regulation of insulin sensitivity, as Sgk1, Sgk2, and Sgk3 triple-knockout mice have similar increases in hepatic insulin sensitivity. In aggregate, these data suggest that targeting hepatic SGK1 may have therapeutic potential in T2D.

CARS senses cysteine deprivation to activate AMPK for cell survival.

Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) is an important cellular metabolite-sensing enzyme that can directly sense changes not only in ATP but also in metabolites associated with carbohydrates and fatty acids. However, less is known about whether and how AMPK senses variations in cellular amino acids. Here, we show that cysteine deficiency significantly triggers calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2)-mediated activation of AMPK. In addition, we found that CaMKK2 directly associates with cysteinyl-tRNA synthetase (CARS), which then binds to AMPKγ2 under cysteine deficiency to activate AMPK. Interestingly, we discovered that cysteine inhibits the binding of CARS to AMPKγ2, and thus, under cysteine deficiency conditions wherein the inhibitory effect of cysteine is abrogated, CARS mediates the binding of AMPK to CaMKK2, resulting in the phosphorylation and activation of AMPK by CaMKK2. Importantly, we demonstrate that blocking AMPK activation leads to cell death under cysteine-deficient conditions. In summary, our study is the first to show that CARS senses the absence of cysteine and activates AMPK through the cysteine-CARS-CaMKK2-AMPKγ2 axis, a novel adaptation strategy for cell survival under nutrient deprivation conditions.

Sulindac and vitamin D3 synergically inhibit proliferation of MCF-7 breast cancer cell through AMPK/Akt/ß-catenin axis in vitro.

Breast cancer is associated with a high rate of recurrence, resistance therapy and mortality worldwide. We aimed at investigating the inhibitory effects of Sulindac and vitamin D3 (VD) on MCF-7 human breast cancer cells. MCF-7 cells were cultured with different concentrations of Sulindac and VD over a period of 24, 48 and 72 hours for cell viability and IC50 experiments. Hochst staining was used to evaluate apoptosis, whereas quantitative PCR (qPCR) was performed to measure mRNA levels of BCL-2 and BAX genes. Immunofluorescence staining was used to monitor intracellular ß-catenin expression. The protein levels of AKT, AMPK and P65 were measured by western blotting. The result showed that cell viability decreased in treated cells dose/time dependently (P < .05). Hochst staining showed an increase in fragmented nuclei in treated cells. The expression of BCL-2 and BAX genes decreased and increased in treated cells, respectively (P < .05). Immunofluorescence staining indicated that the expression of ß-catenin significantly reduced in treated cells. The AKT-1/p-Akt-1 and AMPK/p-AMPK ratio increased in treated cells (P < .05), but the P65/p-P65 ratio did not change significantly (P > .05). Our results indicated that the combination of Sulindac and VD has a growth-inhibiting effect on MCF-7 cells through AMPK/Akt/ß-catenin axis.

Investigation of the specificity and mechanism of action of the ULK1/AMPK inhibitor SBI-0206965.

SBI-0206965, originally identified as an inhibitor of the autophagy initiator kinase ULK1, has recently been reported as a more potent and selective AMP-activated protein kinase (AMPK) inhibitor relative to the widely used, but promiscuous inhibitor Compound C/Dorsomorphin. Here, we studied the effects of SBI-0206965 on AMPK signalling and metabolic readouts in multiple cell types, including hepatocytes, skeletal muscle cells and adipocytes. We observed SBI-0206965 dose dependently attenuated AMPK activator (991)-stimulated ACC phosphorylation and inhibition of lipogenesis in hepatocytes. SBI-0206965 (≥25 µM) modestly inhibited AMPK signalling in C2C12 myotubes, but also inhibited insulin signalling, insulin-mediated/AMPK-independent glucose uptake, and AICA-riboside uptake. We performed an extended screen of SBI-0206965 against a panel of 140 human protein kinases in vitro, which showed SBI-0206965 inhibits several kinases, including members of AMPK-related kinases (NUAK1, MARK3/4), equally or more potently than AMPK or ULK1. This screen, together with molecular modelling, revealed that most SBI-0206965-sensitive kinases contain a large gatekeeper residue with a preference for methionine at this position. We observed that mutation of the gatekeeper methionine to a smaller side chain amino acid (threonine) rendered AMPK and ULK1 resistant to SBI-0206965 inhibition. These results demonstrate that although SBI-0206965 has utility for delineating AMPK or ULK1 signalling and cellular functions, the compound potently inhibits several other kinases and critical cellular functions such as glucose and nucleoside uptake. Our study demonstrates a role for the gatekeeper residue as a determinant of the inhibitor sensitivity and inhibitor-resistant mutant forms could be exploited as potential controls to probe specific cellular effects of SBI-0206965.

PIM kinases inhibit AMPK activation and promote tumorigenicity by phosphorylating LKB1.

BACKGROUND: The oncogenic PIM kinases and the tumor-suppressive LKB1 kinase have both been implicated in the regulation of cell growth and metabolism, albeit in opposite directions. Here we investigated whether these kinases interact with each other to influence AMPK activation and tumorigenic growth of prostate and breast cancer cells. METHODS: We first determined how PIM and LKB1 kinases affect AMPK phosphorylation levels. We then used in vitro kinase assays to demonstrate that LKB1 is phosphorylated by PIM kinases, and site-directed mutagenesis to identify the PIM target sites in LKB1. The cellular functions of PIM and LKB1 kinases were evaluated using either pan-PIM inhibitors or CRISPR/Cas9 genomic editing, with which all three PIM family members and/or LKB1 were knocked out from PC3 prostate and MCF7 breast cancer cell lines. In addition to cell proliferation assays, we examined the effects of PIM and/or LKB1 loss on tumor growth using the chick embryo chorioallantoic membrane (CAM) xenograft model. RESULTS: We provide both genetic and pharmacological evidence to demonstrate that inhibition of PIM expression or activity increases phosphorylation of AMPK at Thr172 in both PC3 and MCF7 cells, but not in their derivatives lacking LKB1. This is explained by our observation that all three PIM family kinases can phosphorylate LKB1 at Ser334. Wild-type LKB1, but not its phosphodeficient derivative, can restore PIM inhibitor-induced AMPK phosphorylation in LKB1 knock-out cells. In the CAM model, loss of LKB1 enhances tumorigenicity of PC3 xenografts, while cells lacking both LKB1 and PIMs exhibit slower proliferation rates and form smaller tumors. CONCLUSION: PIM kinases are novel negative regulators of LKB1 that affect AMPK activity in an LKB1-dependent fashion. The impairment of cell proliferation and tumor growth in cells lacking both LKB1 and PIMs indicates that these kinases possess a shared signaling role in the context of cancer. These data also suggest that PIM inhibitors may be a rational therapeutic option for LKB1-deficient tumors. Video Abstract.

Quercetin protects oral mucosal keratinocytes against lipopolysaccharide-induced inflammatory toxicity by suppressing the AKT/AMPK/mTOR pathway.

BACKGROUND: Cytokines can induce a chronic inflammatory response in the periodontium, leading to periodontitis. Quercetin, a naturally occuring flavonoid, has been shown to inhibit periodontitis, but how it works is poorly understood. In this study, we assessed the impact of quercetin on lipopolysaccharide (LPS)-induced inflammatory damage in oral mucosal keratinocytes (hOMK107) and explored its underlying mechanism. METHODS: The viability and apoptosis of hOMK107 cells were measured after exposure to LPS, followed or not by quercetin. The production of IL-1ß, IL-6, IL-8, TNF-ɑ, iNOS, and COX-2 was quantified by enzyme-linked immunosorbent assay (ELISA), while levels of Akt, AMPK, and mTOR and their phosphorylation were detected semi-quantitatively by western blotting. RESULTS: Quercetin significantly improved cell viability and apoptosis by reversing LPS-induced upregulation of Bax and downregulation of Bcl-2 in hOMK107 cells. Quercetin decreased the production of IL-1ß, IL-6, IL-8, TNF-ɑ, iNOS, and COX-2, as well as signal transduction via the Akt/AMPK/mTOR pathway. Inhibitors of Akt, AMPK, and mTOR strengthened the anti-apoptotic effects of quercetin, while agonists of Akt, AMPK, or mTOR or Akt overexpression weakened the anti-apoptotic effects. CONCLUSION: These results indicate that quercetin may have a potential protective effect against the chronic inflammation-related periodontitis via suppressing Akt/AMPK/mTOR pathway.

miR­351­5p aggravates lipopolysaccharide­induced acute lung injury via inhibiting AMPK.

Inflammation and oxidative stress have indispensable roles in the development of acute lung injury (ALI). MicroRNA (miRNA/miR)­351­5p was initially identified as a myogenesis­associated miRNA; however, its role in lipopolysaccharide (LPS)­induced ALI remains unclear. The aim of the present study was to investigate the role and potential mechanisms of miR­351­5p in ALI. ALI was induced through a single intratracheal injection of LPS for 12 h, and miR­351­5p agomir, antagomir or their corresponding negative controls were injected into the tail vein before LPS stimulation. Compound C, 2',5'­dideoxyadenosine and H89 were used to inhibit AMP­activated protein kinase (AMPK), adenylate cyclase and protein kinase A (PKA), respectively. miR­351­5p levels in the lungs were significantly increased in response to LPS injection. miR­351­5p antagomir alleviated, while miR­351­5p agomir aggravated LPS­induced oxidative stress and inflammation in the lungs. The present results also demonstrated that miR­351­5p antagomir attenuated LPS­induced ALI via activating AMPK, and that the cAMP/PKA axis was required for the activation of AMPK by the miR­351­5p antagomir. In conclusion, the present study indicated that miR­351­5p aggravated LPS­induced ALI via inhibiting AMPK, suggesting that targeting miR­351­5p may help to develop efficient therapeutic approaches for treating ALI.

Diversity-oriented synthesis through gamma radiolysis: Preparation of unusual ecdysteroid derivatives activating Akt and AMPK in skeletal muscle cells.

Gamma-ray radiation is a unique way to induce chemical transformations of bioactive compounds. In the present study, we pursued this approach to the diversity-oriented synthesis of analogs of 20-hydroxyecdysone (20E), an abundant ecdysteroid with a range of beneficial, non-hormonal bioactivities in mammals including humans. Gamma irradiations of aqueous solutions of 20E were conducted either in N2- or N2O-saturated solutions. Centrifugal partition chromatography was used to fractionate crude resulting irradiated materials using a biphasic solvent system composed of tert-butyl alcohol - ethyl acetate - water (0.45:0.9:1, v/v/v) in ascending mode. Subsequently, the products were purified by RP-HPLC. Fourteen ecdysteroids, including five new compounds, were isolated, and their structure were elucidated by 1D and 2D NMR and HRMS. Compounds 2-4, 7, 9, 12 and 15 were tested for their capacity to increase the Akt- and AMPK-phosphorylation of C2C12 murine skeletal myotubes in vitro. The compounds were similarly active on Akt as their parent compound. Stachysterone B (7) and a new ring-rearranged compound (12) were more potent than 20E in activating AMPK, indicating a stronger cytoprotective effect. Our results demonstrate the use of gamma irradiation in expanding the chemical diversity of ecdysteroids to obtain new, unusual bioactive metabolites.

Cereblon contributes to the development of pulmonary fibrosis via inactivation of adenosine monophosphate-activated protein kinase α1.

Pulmonary fibrosis is a progressive and lethal lung disease characterized by the proliferation and differentiation of lung fibroblasts and the accumulation of extracellular matrices. Since pulmonary fibrosis was reported to be associated with adenosine monophosphate-activated protein kinase (AMPK) activation, which is negatively regulated by cereblon (CRBN), we aimed to determine whether CRBN is involved in the development of pulmonary fibrosis. Therefore, we evaluated the role of CRBN in bleomycin (BLM)-induced pulmonary fibrosis in mice and in transforming growth factor-beta 1 (TGF-ß1)-induced differentiation of human lung fibroblasts. BLM-induced fibrosis and the mRNA expression of collagen and fibronectin were increased in the lung tissues of wild-type (WT) mice; however, they were significantly suppressed in Crbn knockout (KO) mice. While the concentrations of TGF-ß1/2 in bronchoalveolar lavage fluid were increased via BLM treatment, they were similar between BLM-treated WT and Crbn KO mice. Knockdown of CRBN suppressed TGF-ß1-induced activation of small mothers against decapentaplegic 3 (SMAD3), and overexpression of CRBN increased it. TGF-ß1-induced activation of SMAD3 increased α-smooth muscle actin (α-SMA) and collagen levels. CRBN was found to be colocalized with AMPKα1 in lung fibroblasts. CRBN overexpression inactivated AMPKα1. When cells were treated with metformin (an AMPK activator), the CRBN-induced activation of SMAD3 and upregulation of α-SMA and collagen expression were significantly suppressed, suggesting that increased TGF-ß1-induced activation of SMAD3 via CRBN overexpression is associated with AMPKα1 inactivation. Taken together, these data suggest that CRBN is a profibrotic regulator and maybe a potential target for treating lung fibrosis.

Mechanism and application of metformin in kidney diseases: An update.

Metformin is an oral antihyperglycemic drug widely used to treat type 2 diabetes mellitus (T2DM), acting via indirect activation of 5' Adenosine monophosphate-activated Protein Kinase (AMPK). Beyond the anti-diabetic effect, accumulative pieces of evidence have revealed that metformin also everts a beneficial effect in diverse kidney diseases. In various acute kidney diseases (AKI) animal models, metformin protects renal tubular cells from inflammation, apoptosis, reactive oxygen stress (ROS), endoplasmic reticulum (ER) stress, epithelial-mesenchymal transition (EMT) via AMPK activation. In diabetic kidney disease (DKD), metformin also alleviates podocyte loss, mesangial cells apoptosis, and tubular cells senescence through AMPK-mediated signaling pathways. Besides, metformin inhibits cystic fibrosis transmembrane conductance regulator (CFTR)-mediated fluids secretion and the mammalian target of rapamycin (mTOR)-involved cyst formation negatively regulated by AMPK in autosomal dominant polycystic kidney disease (APDKD). Furthermore, metformin also contributes to the alleviation of urolithiasis and renal cell carcinoma (RCC). As the common pathway for chronic kidney disease (CKD) progressing towards end-stage renal disease (ESRD), renal fibrosis is ameliorated by metformin, to a great extent dependent on AMPK activation. However, clinical data are not always consistent with preclinical data, some clinical investigations showed the unmeaningful even detrimental effect of metformin on T2DM patients with kidney diseases. Most importantly, metformin-associated lactic acidosis (MALA) is a vital issue restricting the application of metformin. Thus, we conclude the application of metformin in kidney diseases and uncover the underlying molecular mechanisms in this review.

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.

Inhibition of AMP-activated protein kinase in respiratory syncytial virus infection activates lipid metabolism.

Respiratory syncytial virus (RSV) is most commonly associated with upper respiratory tract infections during childhood. The lipid composition of cells and lipogenic enzymes play an important role in RSV infection. There are controversial data about whether lipid biosynthesis regulators such as AMP-activated protein kinase (AMPK) are deregulated by RSV. Hence, we examined whether the activation state of AMPK is altered in RSV-infected HEp-2 cells. Our data show that RSV infection inhibits AMPK activity, favoring the activation of downstream lipogenic effectors and cellular lipid anabolism in HEp-2 cells.

Salinomycin suppresses TGF-ß1-induced EMT by down-regulating MMP-2 and MMP-9 via the AMPK/SIRT1 pathway in non-small cell lung cancer.

Salinomycin (Sal) is a recently identified anti-tumor drug for treating several types of solid tumor; however, its effects on the migratory and invasive properties of non-small cell lung cancer (NSCLC) remain unclear. This study investigated the inhibitory effect underlying mechanisms of Salon transforming growth factor-ß1 (TGF-ß1)-induced epithelial-to-mesenchymal transition (EMT) and cell migration. Sal solidly blocked cell migration and invasion enhancement by TGF-ß1-induced EMT, through recovering E-cadherin loss and suppressing mesenchymal markers induction, as well as TGF-ß1-mediated AMPK/SIRT signaling activity upregulation. The pharmacologic inhibition or knockdown of AMPK or SIRT1 can act synergistically with Sal to inhibit TGF-ß1-induced MMP-2 and MMP-9. In contrast, AMPK or SIRT1 upregulation can protect against TGF-ß1-induced MMP-2 and MMP-9 inhibition by Sal. Next we demonstrated that the MMP-2 and MMP-9 knockdown can act synergistically with Sal to inhibit TGF-ß1-induced EMT. Moreover, treatment of PMA of MMP activator increased TGF-ß1-induced MMP-2 and MMP-9, even with Sal. Our results demonstrate that Sal suppresses TGF-ß1-induced EMT by downregulating MMP-2 and MMP-9 through the AMPK/SIRT pathway, thereby inhibiting lung cancer cell migration and invasion.