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Osteoporosis (OP) is a highly prevalent disorder characterized by low bone mass that severely reduces patient quality of life. Although numerous treatments for OP have been introduced in clinic, many have side effects and high costs. Therefore, there is still an unmet need for optimal solutions. Here, raw signal analysis was used to identify potential high-risk factors for OP, and the biological functions and possible mechanisms of action (MOAs) of these factors were explored via gene set enrichment analysis (GSEA). Subsequently, molecular biological experiments were performed to verify and analyze the discovered risk factors in vitro and in vivo. PMAIP1 was identified as a potential risk factor for OP and significantly suppressed autophagy in osteoblasts via the AMPK/mTOR pathway, thereby inhibiting the proliferation and differentiation of osteoblasts. Furthermore, we constructed an ovariectomy (OVX) model of OP in rats and simultaneously applied si-PMAIP1 for in vivo interference. si-PMAIP1 upregulated the expression of LC3B and p-AMPK and downregulated the expression of p-mTOR, and these effects were reversed by the autophagy inhibitor. Micro-CT revealed that, si-PMAIP1 significantly inhibited the development of osteoporosis in OVX model rats, and this therapeutic effect was attenuated by treatment with an autophagy inhibitor. This study explored the role and mechanism of PMAIP1 in OP and demonstrated that PMAIP1 may serve as a novel target for OP treatment., (© 2024. The Author(s) under exclusive licence to Japan Human Cell Society.)

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.

Objective: To investigate whether thermogenesis and the hypothalamus may be involved in the physiopathology of experimental arthritis (EA)., Methods: EA was induced in male Lewis rats by intradermal injection of Freund's complete adjuvant (CFA). Food intake, body weight, plasma cytokines, thermographic analysis, gene and protein expression of thermogenic markers in brown adipose tissue (BAT) and white adipose tissue (WAT), and hypothalamic AMP-activated protein kinase (AMPK) were analyzed. Virogenetic activation of hypothalamic AMPK was performed., Results: We first demonstrated that EA was associated with increased BAT thermogenesis and browning of subcutaneous WAT leading to elevated energy expenditure. Moreover, rats experiencing EA showed inhibition of hypothalamic AMPK, a canonical energy sensor modulating energy homeostasis at the central level. Notably, specific genetic activation of AMPK in the ventromedial nucleus of the hypothalamus (a key site modulating energy metabolism) reversed the effect of EA on energy balance, brown fat, and browning, as well as promoting amelioration of synovial inflammation in experimental arthritis., Conclusion: Overall, these data indicate that EA promotes a central catabolic state that can be targeted and reversed by the activation of hypothalamic AMPK. This might provide new therapeutic alternatives to treat rheumatoid arthritis (RA)-associated metabolic comorbidities, improving the overall prognosis in patients with RA., (© 2021 The Authors. Arthritis & Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

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., (Copyright © 2021. Published by Elsevier GmbH.)

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., Competing Interests: Conflict of interest None., (Published by Elsevier Ltd.)

AMP-activated protein kinase (AMPK) has an important role in cellular energy homeostasis and has emerged as a promising target for treatment of Type 2 Diabetes (T2D) due to its beneficial effects on insulin sensitivity and glucose homeostasis. O304 is a pan-AMPK activator that has been shown to improve glucose homeostasis in both mouse models of diabetes and in human T2D subjects. Here, we describe the genome-wide transcriptional profile and chromatin landscape of pancreatic islets following O304 treatment of mice fed high-fat diet (HFD). O304 largely prevented genome-wide gene expression changes associated with HFD feeding in CBA mice and these changes were associated with remodelling of active and repressive chromatin marks. In particular, the increased expression of the β-cell stress marker Aldh1a3 in islets from HFD-mice is completely abrogated following O304 treatment, which is accompanied by loss of active chromatin marks in the promoter as well as distant non-coding regions upstream of the Aldh1a3 gene. Moreover, O304 treatment restored dysfunctional glucose homeostasis as well as expression of key markers associated with β-cell function in mice with already established obesity. Our findings provide preclinical evidence that O304 is a promising therapeutic compound not only for T2D remission but also for restoration of β-cell function following remission of T2D diabetes., (© 2021. The Author(s).)

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. Nkx2.1 Cre ; Lkb1 f/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 Nkx2.1 Cre ; Lkb1 f/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 Nkx2.1 Cre ; Lkb1 f/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., (© 2021. The Author(s).)

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)-1 β , 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 (PPAR γ ) and PPAR γ coactivator-1 α 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., Competing Interests: All authors declare that there are no actual or potential conflicts of interest, including any financial, personal, or relationships with other people or organizations that could inappropriately influence or be perceived to influence our work., (Copyright © 2021 Mingchao Li et al.)

To reshape neuronal connectivity in adult stages, Drosophila sensory neurons prune their dendrites during metamorphosis using a genetic degeneration program that is induced by the steroid hormone ecdysone. Metamorphosis is a nonfeeding stage that imposes metabolic constraints on development. We find that AMP-activated protein kinase (AMPK), a regulator of energy homeostasis, is cell-autonomously required for dendrite pruning. AMPK is activated by ecdysone and promotes oxidative phosphorylation and pyruvate usage, likely to enable neurons to use noncarbohydrate metabolites such as amino acids for energy production. Loss of AMPK or mitochondrial deficiency causes specific defects in pruning factor translation and the ubiquitin-proteasome system. Our findings distinguish pruning from pathological neurite degeneration, which is often induced by defects in energy production, and highlight how metabolism is adapted to fit energy-costly developmental transitions., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

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., (©2021 The Authors; Published by the American Association for Cancer Research.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Huang, Liu, Luz, Berrong, Meyer, Zou, Swann, Sundaramoorthy, Kang, Jauhari, Lento, Chao and Racioppi.)

As progressive, chronic, incurable and common reasons for disability and death, neurodegenerative diseases (NDDs) are significant threats to human health. Besides, the increasing prevalence of neuronal gradual degeneration and death during NDDs has made them a global concern. Since yet, no effective treatment has been developed to combat multiple dysregulated pathways/mediators and related complications in NDDs. Therefore, there is an urgent need to create influential and multi-target factors to combat neuronal damages. Accordingly, the plant kingdom has drawn a bright future. Among natural entities, flavonoids are considered a rich source of drug discovery and development with potential biological and medicinal activities. Growing studies have reported multiple dysregulated pathways in NDDs, which among those mediator AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) play critical roles. In this line, critical role of flavonoids in the upregulation of AMPK/PGC-1α pathway seems to pave the road in the treatment of Alzheimer's disease (AD), Parkinson's disease (PD), aging, central nervous system (brain/spinal cord) damages, stroke, and other NDDs. In the present study, the regulatory role of flavonoids in managing various NDDs has been shown to pass through AMPK/PGC-1α signaling pathway., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2021 Wiley-VCH GmbH.)

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 TGFβ 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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors have nothing to disclose., (© The Korean Urological Association, 2021.)

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 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.

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.

l-Theanine is a nonprotein amino acid with much beneficial efficacy. We found that intraperitoneal treatment of the mice with l-theanine (100 mg/kg/day) enhanced adaptive thermogenesis and induced the browning of inguinal white adipose tissue (iWAT) with elevated expression of Prdm16, Ucp1, and other thermogenic genes. Meanwhile, administration of the mice with l-theanine increased energy expenditure. In vitro studies indicated that l-theanine induced the development of brown-like features in adipocytes. The shRNA-mediated depletion of Prdm16 blunted the role of l-theanine in promoting the brown-like phenotypes in adipocytes and in the iWAT of mice. l-theanine treatment enhanced AMPKα phosphorylation both in adipocytes and iWAT. Knockdown of AMPKα abolished l-theanine-induced upregulation of Prdm16 and adipocyte browning. l-Theanine increased the α-ketoglutarate (α-KG) level in adipocytes, which may increase the transcription of Prdm16 by inducing active DNA demethylation on its promoter. AMPK activation was required for l-theanine-induced increase of α-KG and DNA demethylation on the Prdm16 promoter. Moreover, intraperitoneal administration with l-theanine ameliorated obesity, improved glucose tolerance and insulin sensitivity, and reduced plasma triglyceride, total cholesterol, and free fatty acids in the high-fat diet-fed mice. Our results suggest a potential role of l-theanine in combating diet-induced obesity in mice, which may involve l-theanine-induced browning of WAT., (© 2021 by the American Diabetes Association.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Microglia are the resident immune cells of the central nervous system (CNS). It is well established that microglia are activated and polarized to acquire different inflammatory phenotypes, either pro-inflammatory or anti-inflammatory phenotypes, which act as a critical component in the neuroinflammation following intracerebral hemorrhage (ICH). Microglia produce pro-inflammatory mediators at the early stages after ICH onset, anti-inflammatory microglia with neuroprotective effects appear to be suppressed. Previous research found that driving microglia towards an anti-inflammatory phenotype could restrict inflammation and engulf cellular debris. The principal objective of this review is to analyze the phenotypes and dynamic profiles of microglia as well as their shift in functional response following ICH. The results may further the understanding of the body's self-regulatory functions involving microglia following ICH. On this basis, suggestions for future clinical development and research are provided., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Liu, Liu, Wang, Jiang, Bai and Wang.)

Obesity is becoming a pandemic, and its prevalence is still increasing. Considering that obesity increases the risk of developing cardiometabolic diseases, research efforts are focusing on new ways to combat obesity. Brown adipose tissue (BAT) has emerged as a possible target to achieve this for its functional role in energy expenditure by means of increasing thermogenesis. An important metabolic sensor and regulator of whole-body energy balance is AMP-activated protein kinase (AMPK), and its role in energy metabolism is evident. This review highlights the mechanisms of BAT activation and investigates how AMPK can be used as a target for BAT activation. We review compounds and other factors that are able to activate AMPK and further discuss the therapeutic use of AMPK in BAT activation. Extensive research shows that AMPK can be activated by a number of different kinases, such as LKB1, CaMKK, but also small molecules, hormones, and metabolic stresses. AMPK is able to activate BAT by inducing adipogenesis, maintaining mitochondrial homeostasis and inducing browning in white adipose tissue. We conclude that, despite encouraging results, many uncertainties should be clarified before AMPK can be posed as a target for anti-obesity treatment via BAT activation.

Background: Dexmedetomidine (Dex) exerts an effective therapeutic role in numerous diseases associated with ischemia/reperfusion (I/R) injury via its anti-apoptosis properties. Therefore, this study explores the cardioprotective effects of Dex in cardiac microvascular endothelial cells (CMECs) in response to oxygen-glucose deprivation and re-oxygenation (OGD/R) injury and its potential mechanism., Material and Methods: CMECs were pretreatment with different concentration of Dex, then exposed to OGD/R. Cell viability was measured with CCK-8 assay. Apoptosis was evaluated by flow cytometry, and apoptosis-related protein was determined by Western blot. Autophagy was assessed by transmission electron microscopy and autophagy-related proteins. Besides, the role peroxisome proliferator-activated receptors (PPARδ) in Dex-mediated anti-apoptosis property was validated with agonist and antagonist., Results: OGD/R significantly decreased cell viability, increased reactive oxygen species, caused disorder of autophagy, and increased apoptosis in CMECs. Dex enhanced the viability of the OGD/R-treated CMECs and effectively decreased reactive oxygen species production. Autophagy in CMECs was activated by Dex, as evidenced by the increase in the ratio of LC3B-II/I, expression level of Beclin1 and number of autophagosomes in the OGD/R-induced CMECs. The mechanistic investigation indicated that PPARδ antagonist GW501516 aggravated cell damage following OGD/R, while PPARδ agonist GW6471 partly abolished the Dex-mediated protective effects., Conclusions: Dex activated the PPARδ-AMPK-PGC-1α pathway-mediated autophagy in CMECs, therefore to inhibit excessive apoptosis induced by OGD/R. Dex may potentially be a therapeutic intervention for myocardial I/R injury., (© 2020 John Wiley & Sons Ltd.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chin, He, Chow, Yu, Lai and Miaw.)

Caenorhabditis elegans is a model organism widely used for longevity studies. Current advances have been made in the methods that allow automated monitoring of C. elegans behavior. However, ordinary manual assays as well as automated methods have yet to achieve qualitative whole-life analysis of C. elegans longevity based on intrapopulation variation. Here, we utilized live-cell analysis system to determine the parameters of nematode lifespans. Image-based superposition method enabled to determine not only frailty in worms, but also to measure individual and longitudinal lifespan, healthspan, and frailspan. Notably, k-means clustering via principal component analysis revealed four clusters with distinct longevity patterns in wild-type C. elegans. Physiological relevance of clustering was confirmed by assays with pharmacological and/or genetic manipulation of AMP-activated protein kinase (AMPK), a crucial regulator of healthspan. Finally, we focused on W09D10.4 among the possible regulators extracted by integrative expression analysis with existing data sets. Importantly, W09D10.4 knockdown increased the high-healthspan populations only in the presence of AMPK, suggesting that W09D10.4 is a novel AMPK-associated healthspan shortening factor in C. elegans. Overall, the study establishes a novel platform of longitudinal lifespan in C. elegans, which is user-friendly, and may be a useful pharmacological tool to identify healthspan modulatory factors., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

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.

Objective: Dental follicle cells (DFCs) are progenitors of alveolar osteoblasts. AMP-activated protein kinase (AMPK) and the down-stream activated autophagy process play a key role in cellular energy and metabolic homeostasis and are involved in many biological processes including differentiation. Previous studies showed ambiguous results about the role of AMPK and autophagy in osteogenic differentiation of various osteogenic progenitors, but the role of AMPK and autophagy in DFCs is unknown. This study examined the role of AMPK and autophagy in the osteogenic differentiation of DFCs., Materials and Methods: We evaluated the expression of AMPK isoforms and autophagy markers during osteogenic differentiation via Western Blot analyses and the impact of AMPK / autophagy activators and inhibitors and siRNAs on osteogenic differentiation via ALP activity assay, Alizarin Red staining and Real-Time Reverse-Transcription PCR., Results: We have shown that expression of AMPK and autophagy markers are regulated during osteogenic differentiation and that activation of AMPK inhibits the ALP activity and other osteogenic markers after induction of osteogenic differentiation, while inhibition of AMPK and autophagy increased the expression of some osteogenic markers. In long-term cultures with osteogenic differentiation medium, however, both the activation and the inhibition of AMPK significantly inhibited biomineralization of DFCs. In contrast, activation or inhibition of autophagy barely affected early differentiation markers, while autophagy inhibition enhanced biomineralization and autophagy activation diminished mineralization capability of DFCs., Conclusions: AMPK regulates the osteogenic differentiation in earlier stages while indirectly affecting biomineralization at least partly via autophagy. The osteogenic differentiation of DFCs is sensitive to changes in AMPK and autophagic activity., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 ICC‑MSCs. The present study then investigated the role of HVEM‑overexpressing MSCs in the chemoresistance of cholangiocarcinoma cells. It was demonstrated that HVEM‑overexpressing MSCs could support cell survival of chemotherapeutic cholangiocarcinoma cells and inhibited their apoptosis. Further investigations revealed that HVEM‑overexpressing MSCs could secrete IL‑6 and also activated AMPK/mTOR‑dependent autophagy of cholangiocarcinoma cells. Thus, it was concluded that ICC‑MSC‑induced autophagy is the primary cause of chemoresistance in ICC.

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.

AMP-activated protein kinase (AMPK) is the downstream component of a protein kinase cascade that is a key regulator of energy balance at both the cellular and whole-body level. AMPK acts to stimulate ATP production and reduce ATP consumption when cellular ATP levels fall, thereby normalizing energy balance. Given the central role of AMPK in cellular carbohydrate and lipid metabolism, AMPK activation has been proposed to be a therapeutic target for conditions associated with dysfunctional nutrient metabolism including obesity, type 2 diabetes, hepatic steatosis, cardiovascular diseases and cancer. One way by which increased ATP production can be achieved is by increasing the supply of nutrient substrates. In the 1990s, AMPK activation was demonstrated to stimulate glucose uptake in striated muscle, thereby improving substrate supply for ATP production. Subsequently AMPK activation was postulated to underlie the increase in glucose uptake that occurs during muscle contraction. More recently, however, several lines of evidence have demonstrated that AMPK activation is unlikely to be required for contraction-mediated glucose uptake. Furthermore, despite the importance of AMPK in cellular and whole-body metabolism, far fewer studies have investigated either the role of AMPK in glucose uptake by non-muscle tissues or whether AMPK regulates the uptake of fatty acids. In the present review, we discuss the role of AMPK in nutrient uptake by tissues, focusing on glucose uptake out with muscle and fatty acid uptake., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Marein, an active component of the Coreopsis tinctoria Nutt. plant, is known to improve diabetic nephropathy (DN). However, its anti-diabetic functions in DN and potential mechanisms remain unclear. The aim of this study was to elucidate the effects and mechanisms of Marein in diabetic db/db mice with DN, and in high glucose-treated HK-2 cells. In vivo, treating diabetic db/db mice with Marein for 12 consecutive weeks restored diabetes-induced hyperglycemia and dyslipidemia, and ameliorated renal function deterioration, glomerulosclerosis, and renal ectopic lipid deposition. Marein exerted renoprotective effects by directly inhibiting renal tubule sodium glucose transporter 2 (SGLT2) expression, and then activating the AMP-activated protein kinase (AMPK)/acetyl CoA carboxylase (ACC)/peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) pathway in db/db mice. Meanwhile, Marein ameliorated fibrosis and inflammation by suppressing the pro-inflammatory factors interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1), and expression of the extracellular matrix proteins, fibronectin (FN) and collagen 1 (COL1) in diabetic mice. In vitro, MDCK monolayer cells were established to explore the characteristics of Marein transmembrane transport. Marein was found to be absorbed across the membrane at a medium level that involved active transport and this was mediated by SGLTs. In HK-2 cells, Marein decreased uptake of the fluorescent glucose analog, 2-NBDG, by 22 % by inhibiting SGLT2 expression. In high glucose-treated HK-2 cells, Marein decreased SGLT2 expression and increased phosphorylated (p)-AMPK/p-ACC to improve high glucose-induced cellular dysfunction. Furthermore, Marein treatment decreased SGLT2 expression in SGLT2-overexpressing HK-2 cells. In addition, molecular docking and dynamics analysis revealed that SGLT2 was a direct target of Marein. Collectively, our results demonstrated that Marein ameliorates DN by inhibiting renal SGLT2 and activating p-AMPK, suggesting Marein can potentially prevent DN by suppressing renal SGLT2 expression directly., (Copyright © 2020. Published by Elsevier Masson SAS.)

Objective: Glycogen is a major energy reserve in liver and skeletal muscle. The master metabolic regulator AMP-activated protein kinase (AMPK) associates with glycogen via its regulatory β subunit carbohydrate-binding module (CBM). However, the physiological role of AMPK-glycogen binding in energy homeostasis has not been investigated in vivo. This study aimed to determine the physiological consequences of disrupting AMPK-glycogen interactions., Methods: Glycogen binding was disrupted in mice via whole-body knock-in (KI) mutation of either the AMPK β1 (W100A) or β2 (W98A) isoform CBM. Systematic whole-body, tissue and molecular phenotyping was performed in KI and respective wild-type (WT) mice., Results: While β1 W100A KI did not affect whole-body metabolism or exercise capacity, β2 W98A KI mice displayed increased adiposity and impairments in whole-body glucose handling and maximal exercise capacity relative to WT. These KI mutations resulted in reduced total AMPK protein and kinase activity in liver and skeletal muscle of β1 W100A and β2 W98A, respectively, versus WT mice. β1 W100A mice also displayed loss of fasting-induced liver AMPK total and α-specific kinase activation relative to WT. Destabilisation of AMPK was associated with increased fat deposition in β1 W100A liver and β2 W98A skeletal muscle versus WT., Conclusions: These results demonstrate that glycogen binding plays critical roles in stabilising AMPK and maintaining cellular, tissue and whole-body energy homeostasis., (Copyright © 2020 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The Caenorhabditis elegans genome possesses homologs of about two-thirds of all human disease genes. Based on its physiological aging characteristics and superiority, the use of C. elegans as a model system for studies on aging, age-related diseases, mechanisms of longevity, and drug screening has been widely acknowledged in recent decades. Lifespan increasing mutations in C. elegans were found to delay aging by impinging several signaling pathways and related epigenetic modifications, including the insulin/IGF-1 signaling (IIS), AMP-activated protein kinase (AMPK), and mechanistic target of rapamycin (mTOR) pathways. Interestingly, dietary restriction (DR) has been shown to increase the lifespan of numerous metazoans and protect them from multiple age-related pathologies. However, the underlying molecular mechanisms are unclear. In recent decades, C. elegans has been used as a unique model system for high-throughput drug screening. Here, we review C. elegans mutants exhibiting increased in lifespan and age-dependent changes under DR, as well as the utility of C. elegans for drug screening. Thus, we provide evidence for the use of this model organism in research on the prevention of aging., (Copyright © 2020 Zhang, Li, Zhou, Wang and Li.)

Objective: Evidence for AMP-activated protein kinase (AMPK)-mediated regulation of skeletal muscle metabolism during exercise is mainly based on transgenic mouse models with chronic (lifelong) disruption of AMPK function. Findings based on such models are potentially biased by secondary effects related to a chronic lack of AMPK function. To study the direct effect(s) of AMPK on muscle metabolism during exercise, we generated a new mouse model with inducible muscle-specific deletion of AMPKα catalytic subunits in adult mice., Methods: Tamoxifen-inducible and muscle-specific AMPKα1/α2 double KO mice (AMPKα imdKO) were generated by using the Cre/loxP system, with the Cre under the control of the human skeletal muscle actin (HSA) promoter., Results: During treadmill running at the same relative exercise intensity, AMPKα imdKO mice showed greater depletion of muscle ATP, which was associated with accumulation of the deamination product IMP. Muscle-specific deletion of AMPKα in adult mice promptly reduced maximal running speed and muscle glycogen content and was associated with reduced expression of UGP2, a key component of the glycogen synthesis pathway. Muscle mitochondrial respiration, whole-body substrate utilization, and muscle glucose uptake and fatty acid (FA) oxidation during muscle contractile activity remained unaffected by muscle-specific deletion of AMPKα subunits in adult mice., Conclusions: Inducible deletion of AMPKα subunits in adult mice reveals that AMPK is required for maintaining muscle ATP levels and nucleotide balance during exercise but is dispensable for regulating muscle glucose uptake, FA oxidation, and substrate utilization during exercise., (Copyright © 2020 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Mineralocorticoid receptor antagonists (MRAs) are recommended for the treatment of heart failure and hypertension, mainly due to their natriuretic and anti-fibrotic mode of action. Rodent studies have shown that MRAs can prevent adverse metabolic consequences of obesity but an elucidation of underlying molecular mechanisms is missing. Here, we investigated metabolic effects of the novel non-steroidal MRA finerenone (FIN) in a mouse model of high-fat diet (HFD)-induced obesity and the signaling pathways activated by MR antagonism at level of interscapular brown adipose tissue (iBAT). C57BL/6J male mice were fed a normal diet or a HFD (with60% kcal from fat) containing or not FIN for 3 months. Metabolic parameters, adipose tissue morphology, gene and protein expression analysis were assessed. We also used brown adipocyte cultures (T37i cells) to investigate the effects of FIN-mediated MR antagonism upon lipid and mitochondrial metabolism. HFD + FIN-treated mice showed improved glucose tolerance together with increased multilocularity and higher expression of thermogenic markers at the level of iBAT, without differences in white adipose depots, suggesting an iBAT-specific effect of FIN. Mechanistically, FIN increased activation of AMP-activated protein kinase which, in turn, stimulated adipose triglyceride lipase activation, with subsequent increased expression of uncoupling protein-1 in brown adipocytes., (© 2020 Federation of American Societies for Experimental Biology.)

Although previous studies have shown that the administration of fibroblast growth factor 21 (FGF21) reverses hepatic steatosis, the mechanism by which FGF21 exerts a therapeutic effect on nonalcoholic fatty liver disease (NAFLD) is not yet entirely understood. We previously demonstrated that hepatic six transmembrane protein of prostate 2 (STAMP2) may represent a suitable target for NAFLD. We investigated the mechanism underlying the therapeutic effect of recombinant FGF21 on NAFLD, focusing on the involvement of hepatic STAMP2. In this study, we used human nonalcoholic steatosis patient pathology samples, C57BL/6 mice for a high-fat diet (HFD)-induced in vivo NAFLD model, and used human primary hepatocytes and HepG2 cells for oleic acid (OA)-induced in vitro NAFLD model. We observed that recombinant FGF21 treatment ameliorated hepatic steatosis and insulin resistance through the upregulation of STAMP2 expression. We further observed hepatic iron overload (HIO) and reduced iron exporter, ferroportin expression in the liver samples obtained from human NAFLD patients, and HFD-induced NAFLD mice and in OA-treated HepG2 cells. Importantly, recombinant FGF21 improved HIO through the hepatic STAMP2-mediated upregulation of ferroportin expression. Our data suggest that hepatic STAMP2 may represent a suitable therapeutic intervention target for FGF21-induced improvement of NAFLD accompanying HIO., (© 2020 Federation of American Societies for Experimental Biology.)

The tumor microenvironment (TME) and metabolic reprogramming have been implicated in cancer development and progression. However, the link between TME, metabolism, and cancer progression in lung cancer is unclear. In the present study, we identified IMPAD1 from the conditioned medium of highly invasive CL1-5. High expression of IMPAD1 was associated with a poorer clinical phenotype in lung cancer patients, with reduced survival and increased lymph node metastasis. Knockdown of IMPAD1 significantly inhibited migration/invasion abilities and metastasis in vitro and in vivo. Upregulation of IMPAD1 and subsequent accumulation of AMP in cells increased the pAMPK, leading to Notch1 and HEY1 upregulation. As AMP is an ADORA1 agonist, treatment with ADORA1 inhibitor reduced the expression of pAMPK and HEY1 expression in IMPAD1-overexpressing cells. IMPAD1 caused mitochondria dysfunction by inhibiting mitochondrial Complex I activity, which reduced mitochondrial ROS levels and activated the AMPK-HEY1 pathway. Collectively this study supports the multipotent role of IMPAD1 in promotion of lung cancer metastasis by simultaneously increasing AMP levels, inhibition of Complex I activity to decrease ROS levels, thereby activating AMPK-Notch1-HEY1 signaling, and providing an alternative metabolic pathway in energy stress conditions., Competing Interests: Declaration of competing interest No potential conflicts of interest were disclosed., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: Pulmonary hypertension (PH) due to left heart disease (group 2), especially in the setting of heart failure with preserved ejection fraction (HFpEF), is the most common cause of PH worldwide; however, at present, there is no proven effective therapy available for its treatment. PH-HFpEF is associated with insulin resistance and features of metabolic syndrome. The stable prostacyclin analog, treprostinil, is an effective and widely used Food and Drug Administration-approved drug for the treatment of pulmonary arterial hypertension. While the effect of treprostinil on metabolic syndrome is unknown, a recent study suggests that the prostacyclin analog beraprost can improve glucose intolerance and insulin sensitivity. We sought to evaluate the effectiveness of treprostinil in the treatment of metabolic syndrome-associated PH-HFpEF. Approach and Results: Treprostinil treatment was given to mice with mild metabolic syndrome-associated PH-HFpEF induced by high-fat diet and to SU5416/obese ZSF1 rats, a model created by the treatment of rats with a more profound metabolic syndrome due to double leptin receptor defect (obese ZSF1) with a vascular endothelial growth factor receptor blocker SU5416. In high-fat diet-exposed mice, chronic treatment with treprostinil reduced hyperglycemia and pulmonary hypertension. In SU5416/Obese ZSF1 rats, treprostinil improved hyperglycemia with similar efficacy to that of metformin (a first-line drug for type 2 diabetes mellitus); the glucose-lowering effect of treprostinil was further potentiated by the combined treatment with metformin. Early treatment with treprostinil in SU5416/Obese ZSF1 rats lowered pulmonary pressures, and a late treatment with treprostinil together with metformin improved pulmonary artery acceleration time to ejection time ratio and tricuspid annular plane systolic excursion with AMPK (AMP-activated protein kinase) activation in skeletal muscle and the right ventricle., Conclusions: Our data suggest a potential use of treprostinil as an early treatment for mild metabolic syndrome-associated PH-HFpEF and that combined treatment with treprostinil and metformin may improve hyperglycemia and cardiac function in a more severe disease.

Purpose: Various sources of radiation including radiofrequency, electromagnetic radiation (EMR), low- dose X-radiation, low-level microwave radiation and ionizing radiation (IR) are indispensable parts of modern life. In the current review, we discussed the adaptive responses of biological systems to radiation with a focus on the impacts of radiation-induced oxidative stress (RIOS) and its molecular downstream signaling pathways. Materials and methods: A comprehensive search was conducted in Web of Sciences, PubMed, Scopus, Google Scholar, Embase, and Cochrane Library. Keywords included Mesh terms of "radiation," "electromagnetic radiation," "adaptive immunity," "oxidative stress," and "immune checkpoints." Manuscripts published up until December 2019 were included. Results: RIOS induces various molecular adaptors connected with adaptive responses in radiation exposed cells. One of these adaptors includes p53 which promotes various cellular signaling pathways. RIOS also activates the intrinsic apoptotic pathway by depolarization of the mitochondrial membrane potential and activating the caspase apoptotic cascade. RIOS is also involved in radiation-induced proliferative responses through interaction with mitogen-activated protein kinases (MAPks) including p38 MAPK, ERK, and c-Jun N-terminal kinase (JNK). Protein kinase B (Akt)/phosphoinositide 3-kinase (PI3K) signaling pathway has also been reported to be involved in RIOS-induced proliferative responses. Furthermore, RIOS promotes genetic instability by introducing DNA structural and epigenetic alterations, as well as attenuating DNA repair mechanisms. Inflammatory transcription factors including macrophage migration inhibitory factor (MIF), nuclear factor κB (NF-κB), and signal transducer and activator of transcription-3 (STAT-3) paly major role in RIOS-induced inflammation. Conclusion: In conclusion, RIOS considerably contributes to radiation induced adaptive responses. Other possible molecular adaptors modulating RIOS-induced responses are yet to be divulged in future studies.

Zearalenone (ZEA) is a prevalent non-steroidal estrogenic mycotoxin produced mainly by Fusarium contamination. Our previous study showed that ZEA induces the autophagy of Sertoli cells (SCs). However, the underlying mechanisms are still unknown. Several studies have indicated that the increasing level of cytoplasmic Ca 2+ could induce autophagy through CaMKKβ and AMPK pathways. Thus in order to investigate the potential mechanism underlying ZEA-induced autophagy, the activity of calmodulin-dependent kinase kinase β(CaMKKβ)and AMP-activated protein kinase (AMPK) signaling pathway in ZEA-infected TM4 cells was studied. In the present study, ZEA activated the CaMKKβ and AMPK signaling pathways. The AMPK inhibitor and activator significantly inhibited and stimulated the effect of ZEA on AMPK, the transformation from LC3I to LC3II, and the distribution of LC3 dots. In addition, cytosolic calcium (Ca 2+ ) was increased gradually with the concentration of ZEA. After treatment of ZEA-infected cells with 1, 2-bis (2-aminophenoxy) ethane-N, N, N', N'- tetraacetic acid- tetraac etoxymethyl ester (BAPTA-AM) and 2-aminoethyl diphenylborinate (2-APB), the intracellular concentration of Ca 2+ reduced significantly. Also, the activities of CaMKKβ and AMPK and subsequent autophagy decreased. Moreover, the antioxidant NAC significantly decreased activities of AMPK and autophagy -related protein. Therefore, it can be speculated that ROS- mediated ER-stress induced by ZEA activates AMPK via Ca 2+ -CaMKKβ leading to autophagy in TM4 cells., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest associated with this study., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The accurate role of ANRIL in cataract is poorly understood. We aimed to reveal the effects of ANRIL on H 2 O 2 -treated HLECs, SRA01/04, as well as the regulatory mechanisms. Oxidative stress model of HLECs was induced by H 2 O 2 . Cell injury was evaluated according to cell proliferation, apoptosis and DNA damage using CCK-8 assay/flow cytometry and TUNEL assays/γH2AX staining. Expressions of ANRIL and miR-21 in HLECs were determined by RT-qPCR. The effects of miR-21, miR-34a and miR-122-5p inhibition as well as AMPK and β-catenin on HLECs with ANRIL overexpression and H 2 O 2 stimulation were analyzed. In vivo experiment was performed via RT-qPCR. H 2 O 2 repressed proliferation and induced apoptosis or DNA damage in HLECs. Those alterations induced by H 2 O 2 were attenuated by ANRIL overexpression. MiR-21 was positively regulated by ANRIL, and both of them were repressed in H 2 O 2 -induced HLECs and cataract patient tissues. Inhibition of miR-21 but not miR-34a or miR-122-5p reversed the effects of ANRIL on H 2 O 2 -treated HLECs. Phosphorylation of AMPK and expression of β-catenin were increased by ANRIL via regulating miR-21. AMPK and β-catenin affected beneficial function of ANRIL-miR-21 axis.Therefore, lncRNA ANRIL attenuated H 2 O 2 -induced cell injury in HELCs via up-regulating miR-21 via the activation of AMPK and β-catenin.

Background: Mitochondrial oxidative stress plays a prominent role in the development of burn-induced cardiac dysfunction. AMP-activated kinase (AMPK), an energy sensor, has a central role in the pathogenesis of heart failure. However, its role in cardiac dysfunction after burn injury is unclear. Our hypothesis is that burn injury acts through the AMPK-sirtuin 1-PGC1α-nuclear factor erythroid 2-related factor 2 (NFE2L2)-ARE signaling pathway, leading to cardiac mitochondrial impairment, resulting in cardiac dysfunction., Study Design: Male Sprague-Dawley rats underwent sham procedure or 60% total body surface area full-thickness burn. Echocardiograms were performed 24 hours post burn. Heart tissue was harvested at 24 hours post burn for biochemistry/molecular biologic analysis. AC16 cardiomyocytes were treated with either sham or burned rat serum (±AMPK inhibitor/AMPK activator/PGC1α activator) for evaluation of cardiomyocyte mitochondrial function by using seahorse in vitro., Results: Burn injury-induced cardiac dysfunction was measured by echocardiogram. Burn injury suppressed cardiac AMPK, sirtuin 1, and PGC1 expression, leading to acetylation of cardiomyocyte proteins. In addition, burn injury caused NFE2L2 and NFE2L2 regulated antioxidants (heme oxygenase 1, NADH quinone oxidoreductase 1, glutamatecysteine ligase catalytic subunit, manganese superoxide dismutase, and glutathione peroxidase) to decrease, resulting in cardiac oxidative stress. In vitro, AMPK1 activator and PGC1α agonist treatment improved Ac16 cell mitochondrial dysfunction, and AMPK1 inhibitor treatment worsened Ac16 cellular damage., Conclusions: Burn-induced cardiac dysfunction and cardiac mitochondrial damage occur via the AMPK-sirtuin 1-PGC1α-NFE2L2-ARE signaling pathway. AMPK and PGC1α agonists might be promising therapeutic agents to reverse cardiac dysfunction after burn injury., (Copyright © 2020. Published by Elsevier Inc.)

Contraction stimulates skeletal muscle glucose uptake predominantly through activation of AMP-activated protein kinase (AMPK) and Rac1. However, the molecular details of how contraction activates these signaling proteins are not clear. Recently, Axin1 has been shown to form a complex with AMPK and liver kinase B1 during glucose starvation-dependent activation of AMPK. Here, we demonstrate that electrical pulse-stimulated (EPS) contraction of C2C12 myotubes or treadmill exercise of C57BL/6 mice enhanced reciprocal coimmunoprecipitation of Axin1 and AMPK from myotube lysates or gastrocnemius muscle tissue. Interestingly, EPS or exercise upregulated total cellular Axin1 levels in an AMPK-dependent manner in C2C12 myotubes and gastrocnemius mouse muscle, respectively. Also, direct activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleotide treatment of C2C12 myotubes or gastrocnemius muscle elevated Axin1 protein levels. On the other hand, siRNA-mediated Axin1 knockdown lessened activation of AMPK in contracted myotubes. Further, AMPK inhibition with compound C or siRNA-mediated knockdown of AMPK or Axin1 blocked contraction-induced GTP loading of Rac1, p21-activated kinase phosphorylation, and contraction-stimulated glucose uptake. In summary, our results suggest that an AMPK/Axin1-Rac1 signaling pathway mediates contraction-stimulated skeletal muscle glucose uptake.

AMP-activated protein kinase (AMPK) activation promotes early stages of epithelial junction assembly. AMPK activation in MDCK renal epithelial cells facilitates localization of the junction-associated proteins aPKCζ and Par3 to the plasma membrane and promotes conversion of Cdc42, a key regulator of epithelial polarization and junction assembly, to its active GTP bound state. Furthermore, Par3 is an important regulator of AMPK-mediated aPKCζ localization. Both aPKCζ and Par3 serve as intermediates in AMPK-mediated junction assembly, with inhibition of aPKCζ activity or Par3 knockdown disrupting AMPK's ability to facilitate zonula occludens (ZO-1) localization. AMPK phosphorylates the adherens junction protein afadin and regulates its interaction with the tight-junction protein zonula occludens-1. Afadin is phosphorylated at two critical sites, S228 (residing within an aPKCζ consensus site) and S1102 (residing within an AMPK consensus site), that are differentially regulated during junction assembly and that exert different effects on the process. Expression of phospho-defective mutants (S228A and S1102A) perturbed ZO-1 localization to the plasma membrane during AMPK-induced junction assembly. Expression of S228A increased the ZO-1/afadin interaction, while S1102A reduced this interaction during extracellular calcium-induced junction assembly. Inhibition of aPKCζ activity also increased the ZO-1/afadin interaction. Taken together, these data suggest that aPKCζ phosphorylation of afadin terminates the ZO-1/afadin interaction and thus permits the later stages of junction assembly.

Context: Obesity-related insulin resistance (OIR) is one of the main contributors to type 2 diabetes and other metabolic diseases. Protein kinases are implicated in insulin signaling and glucose metabolism. Molecular mechanisms underlying OIR involving global kinase activities remain incompletely understood., Objective: To investigate abnormal kinase activity associated with OIR in human skeletal muscle., Design: Utilization of stable isotopic labeling-based quantitative proteomics combined with affinity-based active enzyme probes to profile in vivo kinase activity in skeletal muscle from lean control (Lean) and OIR participants., Participants: A total of 16 nondiabetic adults, 8 Lean and 8 with OIR, underwent hyperinsulinemic-euglycemic clamp with muscle biopsy., Results: We identified the first active kinome, comprising 54 active protein kinases, in human skeletal muscle. The activities of 23 kinases were different in OIR muscle compared with Lean muscle (11 hyper- and 12 hypo-active), while their protein abundance was the same between the 2 groups. The activities of multiple kinases involved in adenosine monophosphate-activated protein kinase (AMPK) and p38 signaling were lower in OIR compared with Lean. On the contrary, multiple kinases in the c-Jun N-terminal kinase (JNK) signaling pathway exhibited higher activity in OIR vs Lean. The kinase-substrate-prediction based on experimental data further confirmed a potential downregulation of insulin signaling (eg, inhibited phosphorylation of insulin receptor substrate-1 and AKT1/2)., Conclusions: These findings provide a global view of the kinome activity in OIR and Lean muscle, pinpoint novel specific impairment in kinase activities in signaling pathways important for skeletal muscle insulin resistance, and may provide potential drug targets (ie, abnormal kinase activities) to prevent and/or reverse skeletal muscle insulin resistance in humans., (© Endocrine Society 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

High consumption of saturated fats links to the development of hypertension. AMP-activated protein kinase (AMPK), a nutrient-sensing signal, is involved in the pathogenesis of hypertension. We examined whether early intervention with a direct AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR) during pregnancy or lactation can protect adult male offspring against hypertension programmed by high saturated fat consumption via regulation of nutrient sensing signals, nitric oxide (NO) pathway, and oxidative stress. Pregnant Sprague-Dawley rats received regular chow or high saturated fat diet (HFD) throughout pregnancy and lactation. AICAR treatment was introduced by intraperitoneal injection at 50 mg/kg twice a day for 3 weeks throughout the pregnancy period (AICAR/P) or lactation period (AICAR/L). Male offspring ( n = 7-8/group) were assigned to five groups: control, HFD, AICAR/P, HFD + AICAR/L, and HFD + AICAR/P. Male offspring were killed at 16 weeks of age. HFD caused hypertension and obesity in male adult offspring, which could be prevented by AICAR therapy used either during pregnancy or lactation. As a result, we demonstrated that HFD downregulated AMPK/SIRT1/PGC-1α pathway in offspring kidneys. In contrast, AICAR therapy in pregnancy and, to a greater extent, in lactation activated AMPK signaling pathway. The beneficial effects of AICAR therapy in pregnancy is related to restoration of NO pathway. While AICAR uses in pregnancy and lactation both diminished oxidative stress induced by HFD. Our results highlighted that pharmacological AMPK activation might be a promising strategy to prevent hypertension programmed by excessive consumption of high-fat food., Competing Interests: The authors declare no conflict of interest.

We reported recently that activators of AMP-activated protein kinase (AMPK) slow the rundown of current evoked by the D2 autoreceptor agonist quinpirole in rat substantia nigra compacta (SNC) dopamine neurons. The present study examined the effect of AMPK on current generated by dopamine, which unlike quinpirole, is a substrate for the dopamine transporter (DAT). Using whole-cell patch-clamp, we constructed current-voltage (I-V) plots while superfusing brain slices with dopamine (100 μM) for 25 min. Two minutes after starting superfusion, dopamine evoked a peak current with an average slope conductance of 0.97 nS and an estimated reversal potential (E rev ) of -113 mV, which is near that expected for K + . But after 10 min of superfusion, dopamine-evoked currents had shifted to more depolarized values with a slope conductance of 0.64 nS and an E rev of -83 mV. This inward shift in current was completely blocked by the DAT inhibitor GBR12935. However, an AMPK blocking agent (dorsomorphin) permitted the emergence of inward current despite the continued presence of the DAT inhibitor. When D2 autoreceptors were blocked by sulpiride, I-V plots showed that dopamine evoked an inward current with an estimated slope conductance of 0.45 nS with an E rev of -57 mV. Moreover, this inward current was completely blocked by the trace amine-associated receptor 1 (TAAR1) antagonist EPPTB. These results suggest that dopamine activates a TAAR1-dependent non-selective cation current that is regulated by AMPK., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

Objective: To investigate whether exercise improves outcomes of surgery on fatty liver, and whether pharmacological approaches can substitute exercising programs., Summary of Background Data: Steatosis is the hepatic manifestation of the metabolic syndrome, and decreases the liver's ability to handle inflammatory stress or to regenerate after tissue loss. Exercise activates adenosine monophosphate-activated kinase (AMPK) and mitigates steatosis; however, its impact on ischemia-reperfusion injury and regeneration is unknown., Methods: We used a mouse model of simple, diet-induced steatosis and assessed the impact of exercise on metabolic parameters, ischemia-reperfusion injury and regeneration after hepatectomy. The same parameters were evaluated after treatment of mice with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). Mice on a control diet served as age-matched controls., Results: A 4-week-exercising program reversed steatosis, lowered insulin levels, and improved glucose tolerance. Exercise markedly enhanced the ischemic tolerance and the regenerative capacity of fatty liver. Replacing exercise with AICAR was sufficient to replicate the above benefits. Both exercise and AICAR improved survival after extended hepatectomy in mice challenged with a Western diet, indicating protection from resection-induced liver failure., Conclusions: Exercise efficiently counteracts the metabolic, ischemic, and regenerative deficits of fatty liver. AICAR acts as an exercise mimetic in settings of fatty liver disease, an important finding given the compliance issues associated with exercise. Exercising, or its substitution through AICAR, may provide a feasible strategy to negate the hepatic consequences of energy-rich diet, and has the potential to extend the application of liver surgery if confirmed in humans.