AMP-dependent Protein Kinase Research Articles

Coffee Bioactive N-Methylpyridinium: Unveiling Its Antilipogenic Effects by Targeting De Novo Lipogenesis in Human Hepatocytes.

Type 2 diabetes and nonalcoholic fatty liver diseases (NAFLDs) are promoted by insulin resistance (IR), which alters lipid homeostasis in the liver. This study aims to investigate the effect of N-methylpyridinium (NMP), a bioactive alkaloid of coffee brew, on lipid metabolism in hepatocytes. The effect of NMP in modulating lipid metabolism is evaluated at physiological concentrations in a diabetes cell model represented by HepG2 cells cultured in a high-glucose medium. Hyperglycemia triggers lipid droplet accumulation in cells and enhances the lipogenic gene expression, which is transactivated by sterol regulatory element binding protein-1 (SREBP-1). Lipid droplet accumulation alters the redox status and endoplasmic reticulum (ER) stress, leading to the activation of the unfolded protein response and antioxidative pathways by X-Box Binding Protein 1(XBP-1)/eukaryotic Initiation Factor 2 alpha (eIF2α) Protein Kinase RNA-Like ER Kinase and nuclear factor erythroid 2-related factor 2 (NRF2), respectively. NMP induces the phosphorylation of AMP-dependent protein kinase (AMPK) and acetyl-CoA carboxylase α (ACACA), and improves the redox status and ER homeostasis, essential steps to reduce lipogenesis and lipid droplet accumulation. These results suggest that NMP may be beneficial for the management of T2D and NAFLD by ameliorating the cell oxidative and ER homeostasis and lipid metabolism.

Modified (2′-deoxy)adenosines activate autophagy primarily through AMPK/ULK1-dependent pathway

Autophagy is a conserved self-digestion process, which governs regulated degradation of cellular components. Autophagy is upregulated upon energy shortage sensed by AMP-dependent protein kinase (AMPK). Autophagy activators might be contemplated as therapies for metabolic neurodegenerative diseases and obesity, as well as cancer, considering tumor-suppressive functions of autophagy. Among them, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr), a nucleoside precursor of the active phosphorylated AMP analog, is the most commonly used pharmacological modulator of AMPK activity, despite its multiple reported “off-target” effects. Here, we assessed the autophagy/mitophagy activation ability of a small set of (2′-deoxy)adenosine derivatives and analogs using a fluorescent reporter assay and immunoblotting analysis. The first two leader compounds, 7,8-dihydro-8-oxo-2′-deoxyadenosine and -adenosine, are nucleoside forms of major oxidative DNA and RNA lesions. The third, a derivative of inactive N6-methyladenosine with a metabolizable phosphate-masking group, exhibited the highest activity in the series. These compounds primarily contributed to the activation of AMPK and outperformed AICAr; however, retaining the activity in knockout cell lines for AMPK (ΔAMPK) and its upstream regulator SIRT1 (ΔSIRT1) suggests that AMPK is not a main cellular target. Overall, we confirmed the prospects of searching for autophagy activators among (2′-deoxy)adenosine derivatives and demonstrated the applicability of the phosphate-masking strategy for increasing their efficacy.

AMP dependent protein kinase regulates endothelial heparan sulfate expression in response to an inflammatory stimulus under arterial shear stress

Heparan sulfate (HS) is the most abundant glycosaminoglycan on the vascular endothelium and can regulate endothelial cell morphology and function in response to mechanical stimuli. This study investigated endothelial HS response to an inflammatory stimulus under static and arterial shear stress conditions. Human aortic endothelial cells (HAECs) under static conditions expressed significantly higher HS when treated with an inflammatory stimulus compared to untreated controls. HAECs exposed to an inflammatory stimulus after being conditioned with 10 dyn/cm2 of shear stress for 24 h did not express significantly higher HS compared to untreated controls under flow. To investigate the mechanism underlying this differential endothelial HS expression in response to an inflammatory stimulus under static and shear stress conditions, we hypothesized a shear dependent increase in AMP dependent protein kinase (AMPK) was regulating HS response to the inflammatory stimulus. AMPK inhibition using compound C decreased HAEC HS expression in response to inflammatory stimulus under arterial shear stress, revealing AMPK as a regulator of HS expression. Further investigation is needed to elucidate the mechanistic pathways underlying the interactions between HS and AMPK expression in endothelial cells and how they regulate HAEC inflammatory response.

Investigating ZMP Accumulation in C2C12 Myotubes with AICAR and Prodrug-39

5’Adenosine monophosphate (AMP) is a signaling molecule and a known regulator of enzymes in glycolysis, glycogen breakdown, and may play a role in regulating protein synthesis. It is also a known activator of AMP-dependent protein kinase (AMPK), a key energy sensor within the cell. Activation of AMPK has been linked to the regulation of mitochondrial biogenesis, upregulation of BCAA catabolism, and increased skeletal muscle glucose uptake and fatty acid oxidation. AMPK can be activated pharmacologically with 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR), an adenosine analogue. AICAR enters the cell through adenosine transporters and undergoes phosphorylation by Adenosine Kinase forming AICAR monophosphate (ZMP). Unfortunately, the poor bioavailability of AICAR limits its use pharmacologically. Prodrug-39 (P39) is comprised of ZMP with a 4-nitrophenol group to allow membrane permeability and subsequent ZMP formation without the need of membrane transporters. We investigated the ZMP accumulation in C2C12 myotubes following exposure to different concentrations of P39 or AICAR. Based on preliminary data, we hypothesized that AICAR treatments would be more effective at producing ZMP over short periods of time and when treatment concentrations were high, but that P39 treatments would result in greater and more consistent ZMP accumulation over longer periods of time when treatment concentrations were low. We first measured the absorbance of 4-nitrophenol (4NP) within the cell media to determine phosphodiesterase cleavage of P39. Nitrophenol absorbance was 3.9 times higher at 1000 μM compared to 100 μM treatment of P39 after 15 minute incubation, and 13.3 times higher at 11 hours. The nitrophenol absorbance per hour increased linearly between 250 μM to 1000 μM of P39. Adenine nucleotide content and ZMP accumulation in treated cells was determined following organic protein extraction and UV-Vis ultra-performance liquid chromatography (UPLC). After 13 hours of treatment with 500 μM AICAR or P39, ZMP accumulation was 14 fold higher following AICAR treatment compared to P39 (59.7±5.0 vs 4.2±0.1 μmol/g protein). Interestingly, ZMP accumulation was not different between 100 μM treatments (5.6±1.3 vs. 5.7±1.5 μmol/g protein). This suggests that phosphodiesterase cleavage of P39 is likely the limiting factor for ZMP accumulation. Interestingly, while ZMP accumulation following 13 hour incubation with 100 μM AICAR or P39 was not different, ATP content was approximately 50% higher in P39 treated cells (33.7±5.8 vs. 51.0±5.8 μmol/g protein). While the measurement of ZMP accumulation at very low treatment concentrations (10 μM) is very diffcult, P39 exposure for 1 to 1.5 hours resulted in a 3 to 8 fold increase in ZMP content compared to AICAR. This data illustrates the potential for P39 as a drug capable of accumulating greater ZMP than AICAR at very low treatment concentrations, however the capacity of phosphodiesterase activity may limit the application of this drug. This work was funded by a grant from Skylark Biosciences. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The impact of exercise on cardiovascular system: Molecular signaling pathway and cardiac adaptations

The purpose of this review is to describe the impact of endurance and strength physical training on the cardiovascular system by reviewing the molecular signaling pathways, which plays a key role in different muscle adaptations, and the cardiac changes in terms of metabolic and cardiac remodeling, and hemodynamics. In response to endurance-exercise, multiple signaling pathways, including Ca<sup>2+</sup>-dependent pathways, reactive oxygen species (ROS), AMP-dependent protein kinase (AMPK), and mitogen activated protein kinases (p38 MAPK), are involved in the regulation of peroxisome-proliferator-activated receptor-γ coactivator-1α (PGC-1α), which controls the mitochondrial biogenesis. Strength training increases the insulin-like growth factor (IGF-1) which initiates the phosphatidylinositol 3-kinase (PI3-k)-(AKT)-(mTOR) signaling cascade, resulting in the synthesis of proteins and the muscle hypertrophy. In addition to the well-documented changes in skeletal muscle, a critical component of the response to exercise training is the dynamic cardiac remodeling, which is classified as either pathological or physiological depending on triggers.

Protective Effects of NaHS/miR-133a-3p on Lipopolysaccharide-Induced Cardiomyocytes Injury.

The aim of this study was to investigate the effects of sodium hydrosulfide (NaHS) on Lipopolysaccharide (LPS)-induced cardiomyocyte injury in H9c2 cells. H9c2 cardiomyocytes cultivated with medium containing 10 μg/mL LPS were used to recapitulate the phenotypes of those in sepsis. Two sequential experiments were performed. The first contained a control group, a LPS group, and a LPS + NaHS group, with the aim to assure the protective effects of NaHS on LPS-treated cardiomyocytes. The second experiment added a fourth group, the LPS + NaHS + miR-133a-3p inhibition group, with the aim to preliminarily explore whether miR-133-3p exerts a protective function downstream of NaHS. The adenosine triphosphate (ATP) kit was used to detect ATP content; real-time quantitative polynucleotide chain reaction (qPCR) was used to measure the levels of mammalian targets of rapamycin (mTOR), AMP-dependent protein kinase (AMPK), and miR-133a-3p, and Western blot (WB) was used to detect protein levels of mTOR, AMPK, myosin-like Bcl2 interacting protein (Beclin-1), microtubule-associated protein 1 light chain 3 (LC3I/II), and P62 (sequestosome-1, sqstm-1/P62). Compared with the control group, the expressions of miR-133a-3p (P < 0.001), P62 (P < 0.001), and the content of ATP (P < 0.001) decreased, while the expressions of Beclin-1 (P = 0.023) and LC3I/II (P = 0.048) increased in the LPS group. Compared with the LPS group, the expressions of miR-133a-3p (P < 0.001), P62 (P < 0.001), and the content of ATP (P < 0.001) in the NaHS + LPS group increased, while the expressions of Beclin-1 (P = 0.023) and LC3I/II (P = 0.022) decreased. Compared with the NaHS + LPS group, the expression levels of miR-133a-3p (P < 0.001), P62 (P = 0.001), and the content of ATP (P < 0.001) in the LPS + NaHS + miR-133a-3p inhibition group were downregulated, and the expression levels of Beclin-1 (P = 0.012) and LC3I/II (P = 0.010) were upregulated. The difference was statistically significant. There was no significant difference in the expression of AMPK and mTOR between groups. Our research demonstrated that NaHS relieved LPS-induced myocardial injury in H9c2 by promoting the expression of miR-133a-3p, inhibiting autophagy in cardiomyocytes, and restoring cellular ATP levels.

Metabolic control of mitophagy.

Mitochondrial dysfunction is a major hallmark of ageing and related chronic disorders. Controlled removal of damaged mitochondria by the autophagic machinery, a process known as mitophagy, is vital for mitochondrial homeostasis and cell survival. The central role of mitochondria in cellular metabolism places mitochondrial removal at the interface of key metabolic pathways affecting the biosynthesis or catabolism of acetyl-coenzyme A, nicotinamide adenine dinucleotide, polyamines, as well as fatty acids and amino acids. Molecular switches that integrate the metabolic status of the cell, like AMP-dependent protein kinase, protein kinase A, mechanistic target of rapamycin and sirtuins, have also emerged as important regulators of mitophagy. In this review, we discuss how metabolic regulation intersects with mitophagy. We place special emphasis on the metabolic regulatory circuits that may be therapeutically targeted to delay ageing and mitochondria-associated chronic diseases. Moreover, we identify outstanding knowledge gaps, such as the ill-defined distinction between basal and damage-induced mitophagy, which must be resolved to boost progress in this area.

Research progress on AMPK activation in nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) occurrence and progression are associated with lipid accumulation, insulin resistance, inflammation, liver damage, fibrosis, and other factors. AMP-dependent protein kinase (AMPK) is a key molecule that regulates bioenergy metabolism and participates in multiple biological processes, including lipid metabolism, autophagy, inflammation, and cell apoptosis. Promoting AMPK activation can reduce hepatic lipid accumulation and insulin resistance, alleviate the development of NAFLD, reduce liver inflammation and fibrosis, and inhibit the progression of NAFLD to nonalcoholic steatohepatitis.

Two Hormones: Ghrelin and Leptin, Based on AMPK Signaling Pathway, Play a Role in Body Mass Control of Eothenomys miletus during Fasting in Kunming and Dali Regions

Background: The ability to respond to global change and coexist with other species depends on phenotypic plasticity and physiological adaptation techniques of the same species living in various places differ according to the region. Methods: Eothenomys miletus from Kunming (KM) and Dali (DL) under fasting and refeeding acclimation, we examined the thermogenic properties and the associated physiological indicators in the AMP dependent protein kinase (AMPK) pathway. Result: The results demonstrated that energy consumption in E. miletus was decreased by fasting and that the process of survival adaption was significantly influenced by body mass, ghrelin concentration and AMPK activity. Following refeeding, pertinent physiological markers leveled off in the control group, demonstrating the high phenotypic plasticity of E. miletus and the critical role that leptin, ghrelin and AMPK pathways play in energy metabolism and environmental adaption during food fasting. Moreover, there may be a connection between geographical variations in physiological indicators under fasting conditions and variations in the ambient temperature and the food available to E. miletus in various places.

Sexual Dimorphism in Cardiometabolic Diseases: The Role of AMPK.

Cardiovascular diseases (CVDs) are the leading cause of mortality and disability among both males and females. The risk of cardiovascular diseases is heightened by the presence of a risk factor cluster of metabolic syndrome, covering obesity and obesity-related cardiometabolic risk factors such as hypertension, glucose, and lipid metabolism dysregulation primarily. Sex hormones contribute to metabolic regulation and make women and men susceptible to obesity development in a different manner, which necessitates sex-specific management. Identifying crucial factors that protect the cardiovascular system is essential to enhance primary and secondary prevention of cardiovascular diseases and should be explicitly studied from the perspective of sex differences. It seems that AMP-dependent protein kinase (AMPK) may be such a factor since it has the protective role of AMPK in the cardiovascular system, has anti-diabetic properties, and is regulated by sex hormones. Those findings highlight the potential cardiometabolic benefits of AMPK, making it an essential factor to consider. Here, we review information about the cross-talk between AMPK and sex hormones as a critical point in cardiometabolic disease development and progression and a target for therapeutic intervention in human disease.

Xianlinglianxiafang Inhibited the growth and metastasis of triple-negative breast cancer via activating PPARγ/AMPK signaling pathway

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by high invasion and metastasis rates. Xian-Ling-Lian-Xia formula (XLLX) is a traditional Chinese medicine prescription widely used in China for treating TNBC. Clinical studies have shown that XLLX significantly reduces the recurrence and metastasis rate of TNBC and improves disease-free survival. However, the potential molecular mechanisms of XLLX on TNBC are not clear yet. Here, we investigated the effects of XLLX on TNBC using a mouse model and tumor cell lines. The results showed that XLLX significantly inhibited the proliferation, migration, and invasion abilities of TNBC cell lines MDA-MB-231 and 4T1 in vitro, induced apoptosis, and regulated the expression of proliferation, apoptosis, and EMT marker proteins in tumor cells. In in vivo experiments, XLLX treatment significantly reduced the progression of TNBC tumors and lung metastasis. Transcriptomics reveals that XLLX treatment significantly enriched differentially expressed genes in the peroxisome proliferator-activated receptor gamma (PPARγ) and AMP-dependent protein kinase (AMPK) signaling pathways. The western blot results confirmed that XLLX significantly upregulated the protein expression of PPARγ and p-AMPK in TNBC cells, tumors, and lung tissues. It is noteworthy that GW9662 (a PPARγ inhibitor) and Compound C (an AMPK inhibitor) partially reversed the anti-proliferation and anti-metastasis effects of XLLX in TNBC cells. Therefore, XLLX may effectively inhibit the growth and metastasis of TNBC by activating the PPARγ/AMPK signaling pathway.

Xiao-Ban-Xia decoction mitigates cisplatin-induced emesis via restoring PINK1/Parkin mediated mitophagy deficiency in a rat pica model

Ethnopharmacological relevanceAs a traditional Chinese anti-emetic formula, Xiao-Ban-Xia decoction (XBXD) was recorded in Golden Chamber, and has promising anti-emetic effect on chemotherapy-induced nausea and vomiting (CINV). Aim of the studyThis study aimed to determine whether the underlying mechanism of XBXD against CINV is correlated to the restoration of cisplatin-induced PINK1/Parkin mediated mitophagy deficiency and mitigation of gastrointestinal inflammation. Materials and methodsThe rat pica model was established by intraperitoneal injection of cisplatin 6 mg/kg. The daily kaolin consumption, food intake and body weight were recorded every 24 h. The pathological damage of gastric antrum and ileum were observed by hematoxylin-eosin staining. The levels of serum reactive oxygen species (ROS), interleukin-1β (IL-1β) and interleukin-1β (IL-18) were detected by ELISA. The expression of microtubule-associated protein 1 light chain 3 (LC3) in gastric antrum and ileum was detected by Immunofluorescence staining. The levels of LC3II, P62/SQSTM1, PTEN-induced putative protein kinases (PINK1), E3 ubiquitin ligase (Parkin), AMP-dependent protein kinases (AMPK), phosphorylated AMPK (p-AMPK), nuclear factor erythroid 2-related factor (Nrf2) and kelch like ECH Associated Protein 1 (Keap1) in gastric antrum and ileum were assayed by western blotting. ResultsAt 24 h and 72 h following cisplatin challenge, XBXD inhibited cisplatin-induced elevation of kaolin consumption, and improved the daily food intake and body weight loss in rats. Cisplatin-induced gastrointestinal histopathological damages were alleviated, and serum levels of ROS, IL-1β and IL-18 increases were mitigated following XBXD treatments. In gastric antrum and ileum, XBXD activated AMPK-Nrf2 signaling pathway and restored cisplatin-induced PINK1/Parkin mediated mitophagy deficiency. ConclusionsXBXD significantly ameliorated CINV in a cisplatin-induced rat pica model. The underlying anti-emetic mechanism of XBXD might be related to the activation of AMPK-Nrf2 signaling pathway and the restoration of cisplatin-induced PINK1/Parkin-mediated mitophagy deficiency in the gastrointestinal tract.

GP-2250, a novel anticancer agent, inhibits the energy metabolism, activates AMP-Kinase and impairs the NF-kB pathway in pancreatic cancer cells.

GP-2250, a novel anticancer agent, severely limits the energy metabolism, as demonstrated by the inhibition of hexokinase 2 and glyceraldehyde-3-phosphate dehydrogenase and a decrease of ATP. Rescue experiments with supplementary pyruvate or oxaloacetate demonstrated that a TCA cycle deficit largely contributed to cytotoxicity. Activation of the energy-deficit sensor, AMP-dependent protein kinase, was associated with increased phosphorylation of acetyl-CoA carboxylase and Raptor, pointing to a possible deficit in the synthesis of fatty acids and proteins as essential cell components. Binding of p65 to DNA was dose-dependently reduced in nuclear lysates. A transcriptional deficit of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) was substantiated by the downregulation of cyclin D1 and of the anti-apoptotic Bcl2, in line with reduction in tumour cell proliferation and induction of apoptosis, respectively. The upregulation of p53 concomitant with an excess of ROS supported apoptosis. Thus, the anticancer activity of GP-2250 is a result of disruption of energy metabolism and inhibition of tumour promotion by NF-κB.

BushenHuoxue decoction suppresses M1 macrophage polarization and prevents LPS induced inflammatory bone loss by activating AMPK pathway

Abnormal bone metabolism and subsequence osteoporotic fractures are common complications of chronic inflammatory diseases. No effective treatment for these bone-related complications is available at present. The chronic inflammatory state in these diseases has been considered as a key factor of bone loss. Therefore, the combination of inflammation inhibition and bone loss suppression may be an important strategy for reducing bone damage associated with inflammatory diseases. Bushen Huoxue Decoction (BSHXD) is a traditional Chinese herbal compound that has demonstrated the ability to improve bone quality and increase bone density. However, the efficacy of BSHXD on inflammatory bone loss and its underlying mechanisms remain unclear. This study aimed to investigate whether BSHXD inhibits inflammatory bone loss in mice and its potential molecular mechanisms. In the present study, the effect of BSHXD on lipopolysaccharide (LPS)-induced M1 polarization of RAW264.7 macrophage and on local inflammatory bone loss model of mouse skull was determined. The results showed that after treating RAW264.7 cells with LPS for 24 h, the expression levels of IL-1β (39.42 ± 3.076 ng/L, p < 0.05), IL-6 (49.24 ± 1.766 mg/L, p < 0.05) and TNF-α (286.3 ± 27.12 ng/L, p < 0.05) were significantly increased. The addition of BSHXD decreased the expression levels of IL-1β, IL-6, and TNF-α to 31.55 ± 1.296 ng/L, 37.94 ± 0.8869 mg/L, and 196.4 ± 25.25 ng/L, respectively (p < 0.05). The results of immunofluorescence staining, Western blotting (WB) and flow cytometry indicated that the proportion of M1 macrophages in RAW264.7 cells treated with BSHXD for 24 h was significantly lower than that in the LPS group (13.36% ± 0.9829% VS 24.80% ± 4.619%, p < 0.05). The evidence from in-vitro experiments showed that the immunomodulatory ability of BSHXD may be associated with the activation of AMP-dependent protein kinase (AMPK) pathway in LPS-treated macrophages. In addition, the results of micro-CT, H&E staining, immunohistochemical staining and immunofluorescence staining of mouse skull further demonstrated that BSHXD treatment significantly alleviated LPS-induced local bone loss and inflammatory damage in mouse skull model. All results indicated that BSHXD significantly inhibited inflammatory factors release and M1 polarization of macrophage through AMPK signaling pathway. Therefore, BSHXD may be a promising drug for the treatment of inflammatory bone loss.

Accumulation of high-energy phosphates blocks the expression of mitochondrial biogenesis markers and slow-type myosin in soleus muscle under 24 hours of rat hindlimb suspension

Under the initial stage of muscle mechanical unloading, the skeletal muscle undergo accumulation of high-energy phosphates followed by AMP-dependent proteinkinase (AMPK) inactivation. Since AMPK is known to activate mitochondrial biogenesis, it cannot be excluded that AMPK inactivation results in oxidative potential decrease at the later stages of muscle unloading. We decided to test the role of the accumulation of high-energy phosphates in skeletal muscle fibers in the inactivation of mitochondrial biogenesis regulators at an early stage of muscle unloading. To reduce the ATP/ADP ratio, we used beta-guanidine propionic acid, and the obtained data indicating that already during the first day of simulated microgravity, the accumulation of high-energy phosphates can reduce the expression level of mRNA of the key regulator of mitochondrial biogenesis PGC-1α, the transcription factor TFAM, as well as the mitochondrial fusion regulator - mitofusin-1. A number of other parameters of mitochondrial signaling were not subject to changes at this time-point. Thus, we demonstrated the role of the ATP/ADP ratio in the inactivation of several regulators of mitochondrial biogenesis in the postural soleus muscle at an early stage of functional unloading.

Nicaraven protects against endotoxemia-induced inflammation and organ injury through modulation of AMPK/Sirt1 signaling in macrophages

Endotoxemia is a disease characterized by systemic inflammatory responses and organ injury caused by lipopolysaccharide (LPS) infection, with high mortality. Nicaraven (AVS), a potent hydroxyl radical scavenger, has been proven to regulate the inflammatory response in tumors. To investigate the protective effects and mechanisms of AVS in endotoxemia, mice were injected intraperitoneally with LPS to induce endotoxemia. AVS treatment significantly decreased the levels of pro-inflammatory cytokines in the serum, reduced neutrophil infiltration, attenuated multiple organ injury, and increased the survival rate in LPS-challenged mice. In the LPS-induced inflammatory model of macrophages, AVS inhibited macrophage activation, suppressed nitric oxide (NO) production, and inhibited the expression and secretion of pro-inflammatory cytokines. Mechanistically, AVS treatment up-regulated silence information regulator transcript-1 (Sirt1) expression in a time- and dose-dependent manner. AVS treatment activated the AMP-dependent protein kinase (AMPK)/Sirt1 signaling pathway and suppressed the activation of nuclear factor kappa B (NF-κB) in macrophages exposed to LPS. However, the anti-inflammatory effects of AVS could be reversed by the AMPK, the Sirt1 inhibitor, or the histone deacetylase inhibitor. We confirmed that the AMPK inhibitor inhibited AVS-mediated AMPK/Sirt1 activation and NF-κB p65 acetylation. These results suggested that AVS alleviated endotoxemia by activating the AMPK/Sirt1 signaling pathway in macrophages.

Homoplantaginin attenuates high glucose-induced vascular endothelial cell apoptosis through promoting autophagy via the AMPK/TFEB pathway.

Vascular endothelial cell (VEC) injury is a key factor in the development of diabetic vascular complications. Homoplantaginin (Hom), one of the main flavonoids from Salvia plebeia R. Br. has been reported to protect VEC. However, its effects and mechanisms against diabetic vascular endothelium remain unclear. Here, the effect of Hom on VEC was assessed using high glucose (HG)-treated human umbilical vein endothelial cells and db/db mice. In vitro, Hom significantly inhibited apoptosis and promoted autophagosome formation and lysosomal function such as lysosomal membrane permeability and the expression of LAMP1 and cathepsin B. The antiapoptosis effect of Hom was reversed by autophagy inhibitor chloroquine phosphate or bafilomycin A1. Furthermore, Hom promoted gene expression and nuclear translocation of transcription factor EB (TFEB). TFEB gene knockdown attenuated the effect of Hom on upregulating lysosomal function and autophagy. Moreover, Hom activated adenosine monophosphate-dependent protein kinase (AMPK) and inhibited the phosphorylation of mTOR, p70S6K, and TFEB. These effects were attenuated by AMPK inhibitor Compound C. Molecular docking showed a good interaction between Hom and AMPK protein. Animal studies indicated that Hom effectively upregulated the protein expression of p-AMPK and TFEB, enhanced autophagy, reduced apoptosis, and alleviated vascular injury. These findings revealed that Hom ameliorated HG-mediated VEC apoptosis by enhancing autophagy via the AMPK/mTORC1/TFEB pathway.

A novel combination of isovanillin, curcumin, and harmine (GZ17-6.02) enhances cell death and alters signaling in actinic keratoses cells when compared to individual components and two-component combinations.

Actinic keratosis is a pre-malignant skin disease caused by excessive exposure to ultraviolet light. The present studies further defined the biology of a novel combination of isovanillin, curcumin, and harmine in actinic keratosis cells in vitro . An oral formulation (GZ17-6.02) and topical preparation (GZ21T) comprised of the same fixed, stoichiometric ratio have been developed. Together, the three active ingredients killed actinic keratosis cells more effectively than any of its component parts as either individual agents or when combined in pairs. The three active ingredients caused greater levels of DNA damage than any of its component parts as either individual agents or when combined in pairs. As a single agent, compared to isolated components, GZ17-6.02/GZ21T caused significantly greater activation of PKR-like endoplasmic reticulum kinase, the AMP-dependent protein kinase, and ULK1 and significantly reduced the activities of mTORC1, AKT, and YAP. Knockdown of the autophagy-regulatory proteins ULK1, Beclin1, or ATG5 significantly reduced the lethality of GZ17-6.02/GZ21T alone. Expression of an activated mammalian target of rapamycin mutant suppressed autophagosome formation and autophagic flux and reduced tumor cell killing. Blockade of both autophagy and death receptor signaling abolished drug-induced actinic keratosis cell death. Our data demonstrate that the unique combination of isovanillin, curcumin, and harmine represents a novel therapeutic with the potential to treat actinic keratosis in a manner different from the individual components or pairs of the components.

Mitochondrial pyruvate carrier inhibition initiates metabolic crosstalk to stimulate branched chain amino acid catabolism

ObjectiveThe mitochondrial pyruvate carrier (MPC) has emerged as a therapeutic target for treating insulin resistance, type 2 diabetes, and nonalcoholic steatohepatitis (NASH). We evaluated whether MPC inhibitors (MPCi) might correct impairments in branched chain amino acid (BCAA) catabolism, which are predictive of developing diabetes and NASH. MethodsCirculating BCAA concentrations were measured in people with NASH and type 2 diabetes, who participated in a recent randomized, placebo-controlled Phase IIB clinical trial to test the efficacy and safety of the MPCi MSDC-0602K (EMMINENCE; NCT02784444). In this 52-week trial, patients were randomly assigned to placebo (n = 94) or 250 mg MSDC-0602K (n = 101). Human hepatoma cell lines and mouse primary hepatocytes were used to test the direct effects of various MPCi on BCAA catabolism in vitro. Lastly, we investigated how hepatocyte-specific deletion of MPC2 affects BCAA metabolism in the liver of obese mice and MSDC-0602K treatment of Zucker diabetic fatty (ZDF) rats. ResultsIn patients with NASH, MSDC-0602K treatment, which led to marked improvements in insulin sensitivity and diabetes, had decreased plasma concentrations of BCAAs compared to baseline while placebo had no effect. The rate-limiting enzyme in BCAA catabolism is the mitochondrial branched chain ketoacid dehydrogenase (BCKDH), which is deactivated by phosphorylation. In multiple human hepatoma cell lines, MPCi markedly reduced BCKDH phosphorylation and stimulated branched chain keto acid catabolism; an effect that required the BCKDH phosphatase PPM1K. Mechanistically, the effects of MPCi were linked to activation of the energy sensing AMP-dependent protein kinase (AMPK) and mechanistic target of rapamycin (mTOR) kinase signaling cascades in vitro. BCKDH phosphorylation was reduced in liver of obese, hepatocyte-specific MPC2 knockout (LS-Mpc2−/−) mice compared to wild-type controls concomitant with activation of mTOR signaling in vivo. Finally, while MSDC-0602K treatment improved glucose homeostasis and increased the concentrations of some BCAA metabolites in ZDF rats, it did not lower plasma BCAA concentrations. ConclusionsThese data demonstrate novel cross talk between mitochondrial pyruvate and BCAA metabolism and suggest that MPC inhibition leads to lower plasma BCAA concentrations and BCKDH phosphorylation by activating the mTOR axis. However, the effects of MPCi on glucose homeostasis may be separable from its effects on BCAA concentrations.

Inhibition of phosphodiesterase 5A by tadalafil improves SIRT1 expression and activity in insulin-resistant podocytes

A decrease in intracellular levels of 3′,5′-cyclic guanosine monophosphate (cGMP) has been implicated in the progression of diabetic nephropathy. Hyperglycemia significantly inhibits cGMP-dependent pathway activity in the kidney, leading to glomerular damage and proteinuria. The enhancement of activity of this pathway that is associated with an elevation of cGMP levels may be achieved by inhibition of the cGMP specific phosphodiesterase 5A (PDE5A) using selective inhibitors, such as tadalafil. Hyperglycemia decreased the insulin responsiveness of podocytes and impaired podocyte function. These effects were associated with lower protein amounts and activity of the protein deacetylase sirtuin 1 (SIRT1) and a decrease in the phosphorylation of adenosine monophosphate-dependent protein kinase (AMPK). We found that PDE5A protein levels increased in hyperglycemia, and PDE5A downregulation improved the insulin responsiveness of podocytes with reestablished SIRT1 expression and activity. PDE5A inhibitors potentiate nitric oxide (NO)/cGMP signaling, and NO modulates the activity and expression of SIRT1. Therefore, we investigated the effects of tadalafil on SIRT1 and AMPK in the context of improving the insulin sensitivity in podocytes and podocyte function in hyperglycemia. Our study revealed that tadalafil restored SIRT1 expression and activity and activated AMPK by increasing its phosphorylation. Tadalafil also restored stimulating effect of insulin on glucose transport in podocytes with high glucose-induced insulin resistance. Additionally, tadalafil improved the function of podocytes that were exposed to high glucose concentrations. Our results display novel mechanisms involved in the pathogenesis of glomerulopathies in diabetes, which may contribute to the development of more effective treatment strategies for diabetic nephropathy.