AMP-activated Protein Kinase Signaling Research Articles

Gentiopicroside enhances the protective effect of trimetazidine against myocardial ischemia-reperfusion injury via the AMPK/NLRP3 inflammasome signaling.

Myocardial ischemia-reperfusion injury (MI/R) leads to the inevitable clinical consequences of myocardial infarction and subsequent heart failure. Trimetazidine (TMZ), an anti-ischemic agent, exerts protective potential in MI/R but had limited efficacy for some patients. Here we sought to investigate the single and combined application of gentiopicroside (GPS) and TMZ in MI/R. Notably, GPS had little cytotoxicity to cardiomyocytes. GPS attenuated hypoxia/reoxygenation (H/R)-induced cell death, reactive oxygen species production, lactate dehydrogenase and malondialdehyde releases, and antioxidant stress enzyme superoxide dismutase activity, indicating the protective efficacy of GPS against H/R-induced oxidative injury. Importantly, GPS enhanced the protective efficacy of TMZ against H/R-mediated cardiomyocyte injury. Additionally, GPS mitigated the transcription and releases of pro-inflammatory cytokine interleukin-6 and tumor necrosis factor-α in H/R-treated cardiomyocytes, which were enhanced after co-treatment with TMZ. Mechanistically, GPS activated the AMP-activated protein kinase (AMPK) signaling to inhibit H/R-induced NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome, which was further enhanced after costimulation with TMZ. Importantly, blocking the AMPK signaling reversed the protective roles of GPS and its combination with TMZ in H/R-induced oxidative insult and inflammation. In vivo, both GPS and TMZ alleviated the abnormal cardiac structure, cardiomyocyte apoptosis, and cardiac dysfunction in MI/R rats, which were further enhanced after administration with GPS and TMZ together. Furthermore, GPS intensified TMZ-mediated inhibition of oxidative injury, inflammation, and the AMPK/NLRP3 signaling in MI/R rats. Collectively, GPS enhances the protective efficacy of TMZ against MI/R injury through AMPK activation-mediated inhibition of NLRP3 inflammasome signaling, implying a promising therapeutic agent for the treatment of MI/R.

Germline stem cell integrity and quiescence are controlled by an AMPK-dependent neuronal trafficking pathway

During periods of energetic stress, Caenorhabditis elegans can execute a developmentally quiescent stage called "dauer", during which all germline stem cells undergo a G2 cell cycle arrest. In animals that lack AMP-activated protein kinase (AMPK) signalling, the germ cells fail to arrest, undergo uncontrolled proliferation, and lose their reproductive capacity upon recovery from this quiescent stage. These germline defects are accompanied by, and likely result from, an altered chromatin landscape and gene expression program. Through genetic analysis we identified an allele of tbc-7, a predicted RabGAP protein that functions in the neurons, which when compromised, suppresses the germline hyperplasia in the dauer larvae, as well as the post-dauer sterility and somatic defects characteristic of AMPK mutants. This mutation also corrects the abundance and aberrant distribution of transcriptionally activating and repressive chromatin marks in animals that otherwise lack all AMPK signalling. We identified RAB-7 as one of the potential RAB proteins that is modulated by tbc-7 and show that the activity of RAB-7 is critical for the maintenance of germ cell integrity during the dauer stage. We reveal that TBC-7 is regulated by AMPK through two mechanisms when the animals enter the dauer stage. Acutely, the AMPK-mediated phosphorylation of TBC-7 reduces its activity, potentially by autoinhibition, thereby preventing the inactivation of RAB-7. In the more long term, AMPK regulates the miRNAs mir-1 and mir-44 to attenuate tbc-7 expression. Consistent with this, animals lacking mir-1 and mir-44 are post-dauer sterile, phenocopying the germline defects of AMPK mutants. Altogether, we have uncovered an AMPK-dependent and microRNA-regulated cellular trafficking pathway that is initiated in the neurons, and is critical to control germline gene expression cell non-autonomously in response to adverse environmental conditions.

Combined transcriptomics and metabolomics analysis reveals lipid metabolic disruption in swamp eel (Monopterus albus) under chronic waterborne copper exposure

Excessive copper can induce many adverse effects although it's an essential trace element in organisms. The effects of copper on the lipid metabolism have aroused increasing attention. This study investigated the liver lipid metabolism in swamp eel (Monopterus albus, M. albus) chronically exposed to 0, 10, 50, and 100 μg/L Cu2+ for 56 days. The results showed that copper increased the contents of triglyceride (TG), total cholesterol (T-CHO), non-esterified fatty acids (NEFA), and lipid droplets. Transcriptomic analysis found 1901 differentially expressed genes (DEGs) and 140 differential alternative splicing (DAS) genes in the 50 μg/L Cu2+ group, and 1787 DEGs and 184 DAS genes in the 100 μg/L Cu2+ group, respectively, which were enriched in peroxisome proliferator-activated receptor (PPAR), adenosine 5‘-monophosphate (AMP)-activated protein kinase (AMPK), and other signaling pathways. The expression levels of key genes related to PPAR and AMPK signaling pathways were significantly down-regulated after chronic exposure to Cu2+. Meanwhile, metabolomics analysis showed that 52 and 110 differentially expressed metabolites (DEMs) were identified, which were mainly enriched in glycerophospholipids metabolism and steroid synthesis. Moreover, combined analysis of transcriptome and metabolome showed that glycerophospholipid metabolism co-enriched 19 down-regulated DEGs and 4 down-regulated DEMs. Taken together, our results suggested that chronic waterborne copper exposure promoted lipid synthesis, disrupted the metabolic homeostasis of glycerophospholipid, and led to excessive hepatic lipid deposition in M. albus. The combined omics approach enhanced our understanding of copper pollution to lipid metabolism.

Metabolic Syndrome and Cardiac Remodeling Due to Mitochondrial Oxidative Stress Involving Gliflozins and Sirtuins.

To address the mechanistic pathways focusing on mitochondria dysfunction, oxidative stress, sirtuins imbalance, and other contributors in patient with metabolic syndrome and cardiovascular disease. Sodium glucose co-transporter type 2 (SGLT-2) inhibitors deeply influence these mechanisms. Recent randomized clinical trials have shown impressive results in improving cardiac function and reducing cardiovascular and renal events. These unexpected results generate the need to deepen our understanding of the molecular mechanisms able to generate these effects to help explain such significant clinical outcomes. Cardiovascular disease is highly prevalent among individuals with metabolic syndrome and diabetes. Furthermore, mitochondrial dysfunction is a principal player in its development and persistence, including the consequent cardiac remodeling and events. Another central protagonist is the renin-angiotensin system; the high angiotensin II (Ang II) activity fuel oxidative stress and local inflammatory responses. Additionally, sirtuins decline plays a pivotal role in the process; they enhance oxidative stress by regulating adaptive responses to the cellular environment and interacting with Ang II in many circumstances, including cardiac and vascular remodeling, inflammation, and fibrosis. Fasting and lower mitochondrial energy generation are conditions that substantially reduce most of the mentioned cardiometabolic syndrome disarrangements. In addition, it increases sirtuins levels, and adenosine monophosphate-activated protein kinase (AMPK) signaling stimulates hypoxia-inducible factor-1β (HIF-1 beta) and favors ketosis. All these effects favor autophagy and mitophagy, clean the cardiac cells with damaged organelles, and reduce oxidative stress and inflammatory response, giving cardiac tissue protection. In this sense, SGLT-2 inhibitors enhance the level of at least four sirtuins, some located in the mitochondria. Moreover, late evidence shows that SLGT-2 inhibitors mimic this protective process, improving mitochondria function, oxidative stress, and inflammation. Considering the previously described protection at the cardiovascular level is necessary to go deeper in the knowledge of the effects of SGLT-2 inhibitors on the mitochondria function. Various of the protective effects these drugs clearly had shown in the trials, and we briefly describe it could depend on sirtuins enhance activity, oxidative stress reduction, inflammatory process attenuation, less interstitial fibrosis, and a consequent better cardiac function. This information could encourage investigating new therapeutic strategies for metabolic syndrome, diabetes, heart and renal failure, and other diseases.

Lactobacillus plantarum postbiotics trigger AMPK-dependent autophagy to suppress Salmonella intracellular infection and NLRP3 inflammasome activation.

We previously found that Lactobacillus plantarum (LP)-derived postbiotics protected animals against Salmonella infection, but the molecular mechanism remains obscure. This study clarified the mechanisms from the perspective of autophagy. Intestinal porcine epithelial cells (IPEC-J2) were pretreated with LP-derived postbiotics (the culture supernatant, LPC; or heat-killed bacteria, LPB), and then challenged with Salmonella enterica Typhimurium (ST). Results showed that LP postbiotics markedly triggered autophagy under ST infection, as indicated by the increased LC3 and Beclin1 and the decreased p62 levels. Meanwhile, LP postbiotics (particularly LPC) exhibited a strong capacity of inhibiting ST adhesion, invasion and replication. Pretreatment with the autophagy inhibitor 3-methyladenine (3-MA) led to a significant decrease of autophagy and the aggravated infection, indicating the importance of autophagy in LP postbiotics-mediated Salmonella elimination. LP postbiotics (especially LPB) significantly suppressed ST-induced inflammation by modulating inflammatory cytokines (the increased interleukin (IL)-4 and IL-10, and decreased tumor necrosis factor-α (TNF), IL-1β, IL-6 and IL-18). Furthermore, LP postbiotics inhibited NOD-like receptor protein 3 (NLRP3)inflammasome activation, as evidenced by the decreased levels of NLRP3, Caspase-1 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). Deficits in autophagy resulted in an increase of inflammatory response and inflammasome activation. Finally, we found that both LPC and LPB triggered AMP-activated protein kinase (AMPK) signaling pathway to induce autophagy, and this was further confirmed by AMPK RNA interference. The intracellular infection and NLRP3 inflammasome were aggravated after AMPK knockdown. In summary, LP postbiotics trigger AMPK-mediated autophagy to suppress Salmonella intracellular infection and NLRP3 inflammasome in IPEC-J2 cells. Our findings highlight the effectiveness of postbiotics, and provide a new strategy for preventing Salmonella infection.

Alpha-SNAP (M105I) mutation promotes neuronal differentiation of neural stem/progenitor cells through overactivation of AMPK.

Background: The M105I point mutation in α-SNAP (Soluble N-ethylmaleimide-sensitive factor attachment protein-alpha) leads in mice to a complex phenotype known as hyh (hydrocephalus with hop gait), characterized by cortical malformation and hydrocephalus, among other neuropathological features. Studies performed by our laboratory and others support that the hyh phenotype is triggered by a primary alteration in embryonic neural stem/progenitor cells (NSPCs) that leads to a disruption of the ventricular and subventricular zones (VZ/SVZ) during the neurogenic period. Besides the canonical role of α-SNAP in SNARE-mediated intracellular membrane fusion dynamics, it also negatively modulates AMP-activated protein kinase (AMPK) activity. AMPK is a conserved metabolic sensor associated with the proliferation/differentiation balance in NSPCs. Methods: Brain samples from hyh mutant mice (hydrocephalus with hop gait) (B6C3Fe-a/a-Napahyh/J) were analyzed by light microscopy, immunofluorescence, and Western blot at different developmental stages. In addition, NSPCs derived from WT and hyh mutant mice were cultured as neurospheres for in vitro characterization and pharmacological assays. BrdU labeling was used to assess proliferative activity in situ and in vitro. Pharmacological modulation of AMPK was performed using Compound C (AMPK inhibitor) and AICAR (AMPK activator). Results: α-SNAP was preferentially expressed in the brain, showing variations in the levels of α-SNAP protein in different brain regions and developmental stages. NSPCs from hyh mice (hyh-NSPCs) displayed reduced levels of α-SNAP and increased levels of phosphorylated AMPKα (pAMPKαThr172), which were associated with a reduction in their proliferative activity and a preferential commitment with the neuronal lineage. Interestingly, pharmacological inhibition of AMPK in hyh-NSPCs increased proliferative activity and completely abolished the increased generation of neurons. Conversely, AICAR-mediated activation of AMPK in WT-NSPCs reduced proliferation and boosted neuronal differentiation. Discussion: Our findings support that α-SNAP regulates AMPK signaling in NSPCs, further modulating their neurogenic capacity. The naturally occurring M105I mutation of α-SNAP provokes an AMPK overactivation in NSPCs, thus connecting the α-SNAP/AMPK axis with the etiopathogenesis and neuropathology of the hyh phenotype.

Effect and mechanism of Magnolia officinalis pharmacopuncture for treating localized fat via network pharmacology and experimental study

BackgroundRecently, for various reasons, the need for non-invasive treatment for localized fat has emerged. This study confirmed whether Magnolia officinalis (MO) pharmacopuncture reduces localized fat by promoting lipolysis and inhibiting adipogenesis. MethodsThe network was built using genes related to the active compound of MO and the mode of action of MO was predicted by the functional enrichment analysis. Based on the result from network analysis, 100 µL of 2 mg/mL MO pharmacopuncture was injected into the inguinal fat pad for 6 weeks in obese C57BL/6J mice. Normal saline was injected into the right-side inguinal fat pad as a self-control. ResultsIt was expected that the AMP-activated protein kinase (AMPK) signaling pathway would be affected by the MO Network. MO pharmacopuncture reduced the weight and size of inguinal fat in HFD-induced obese mice. The phosphorylation of AMPK along with the increases of lipases was significantly increased by MO injection. Also, the expression levels of fatty acid synthesize-related mediators were suppressed by MO injection. ConclusionOur results demonstrated that MO pharmacopuncture promoted the expression of AMPK, which has beneficial effects on activation of lipolysis and inhibition of lipogenesis. Pharmacopuncture of MO can be a non-surgical alternative therapy in the treatment of local fat tissue.

Integrative metabolomic and transcriptomic analysis reveals difference in glucose and lipid metabolism in the longissimus muscle of Luchuan and Duroc pigs.

Luchuan pig, an obese indigenous Chinese porcine breed, has a desirable meat quality and reproductive capacity. Duroc, a traditional western breed, shows a faster growth rate, high feed efficiency and high lean meat rate. Given the unique features these two porcine breeds have, it is of interest to investigate the underlying molecular mechanisms behind their distinctive nature. In this study, the metabolic and transcriptomic profiles of longissimus dorsi muscle from Duroc and Luchuan pigs were compared. A total of 609 metabolites were identified, 77 of which were significantly decreased in Luchuan compared to Duroc, and 71 of which were significantly elevated. Most differentially accumulated metabolites (DAMs) upregulated in Luchuan were glycerophospholipids, fatty acids, oxidized lipids, alcohols, and amines, while metabolites downregulated in Luchuan were mostly amino acids, organic acids and nucleic acids, bile acids and hormones. From our RNA-sequencing (RNA-seq) data we identified a total of 3638 differentially expressed genes (DEGs), 1802 upregulated and 1836 downregulated in Luchuan skeletal muscle compared to Duroc. Combined multivariate and pathway enrichment analyses of metabolome and transcriptome results revealed that many of the DEGs and DAMs are associated with critical energy metabolic pathways, especially those related to glucose and lipid metabolism. We examined the expression of important DEGs in two pathways, AMP-activated protein kinase (AMPK) signaling pathway and fructose and mannose metabolism, using Real-Time Quantitative Reverse Transcription PCR (qRT-PCR). Genes related to glucose uptake, glycolysis, glycogen synthesis, fatty acid synthesis (PFKFB1, PFKFB4, MPI, TPI1, GYS1, SLC2A4, FASN, IRS1, ULK1) are more activated in Luchuan, while genes related to fatty acid oxidation, cholesterol synthesis (CPT1A, HMGCR, FOXO3) are more suppressed. Energy utilization can be a decisive factor to the distinctive metabolic, physiological and nutritional characteristics in skeletal muscle of the two breeds we studied. Our research may facilitate future porcine breeding projects and can be used to reveal the potential molecular basis of differences in complex traits between various breeds.

Acute Kidney Injury Induces Oxidative Stress and Hepatic Lipid Accumulation through AMPK Signaling Pathway.

Acute kidney injury (AKI) often impairs the function of other organs leading to distant organ injury. The liver is the major organ that regulates metabolism and lipid homeostasis in the body. It has been reported that AKI causes liver injury with increased oxidative stress, inflammatory response and steatosis. In the present study, we investigated the mechanisms by which ischemia-reperfusion-induced AKI caused hepatic lipid accumulation. Kidney ischemia (45 min)-reperfusion (24 h) led to a significant increase in plasma creatinine and transaminase in Sprague Dawley rats, indicating kidney and liver injury. Histological and biochemical analyses revealed hepatic lipid accumulation with a significant elevation of triglyceride and cholesterol levels in the liver. This was accompanied by a decreased AMP-activated protein kinase (AMPK) phosphorylation, indicating the reduced activation of AMPK, which is an energy sensor that regulates lipid metabolism. The expression of AMPK-regulated genes that were responsible for fatty acid oxidation (CPTIα, ACOX) was significantly decreased, while the expression of lipogenesis genes (SREPB-1c, ACC1) was significantly elevated. The oxidative stress biomarker malondialdehyde was elevated in the plasma and liver. Incubation of HepG2 cells with an oxidative stress inducer hydrogen peroxide inhibited AMPK phosphorylation and caused cellular lipid accumulation. This was accompanied by decreased expression of genes responsible for fatty acid oxidation and increased expression of genes responsible for lipogenesis. These results suggest that AKI elicits hepatic lipid accumulation through decreased fatty acid metabolism and increased lipogenesis. Oxidative stress may contribute, in part, to the downregulation of the AMPK signaling pathway leading to hepatic lipid accumulation and injury.

Discovery of IHMT-MST1-39 as a novel MST1 kinase inhibitor and AMPK activator for the treatment of diabetes mellitus

Insulin-producing pancreatic β cell death is the fundamental cause of type 1 diabetes (T1D) and a contributing factor to type 2 diabetes (T2D). Moreover, metabolic disorder is another hallmark of T2D. Mammalian sterile 20-like kinase 1 (MST1) contributes to the progression of diabetes mellitus through apoptosis induction and acceleration of pancreatic β cell dysfunction. AMP-activated protein kinase (AMPK) is an energy sensing kinase and its activation has been suggested as a treatment option for metabolic diseases. Thus, pharmacological inhibition of MST1 and activation of AMPK simultaneously represents a promising approach for diabetes therapy. Here, we discovered a novel selective MST1 kinase inhibitor IHMT-MST1-39, which exhibits anti-apoptosis efficacy and improves the survival of pancreatic β cells under diabetogenic conditions, as well as primary pancreatic islets in an ex vivo disease model. Mechanistically, IHMT-MST1-39 activated AMPK signaling pathway in hepatocytes in vitro, combination of IHMT-MST1-39 and metformin synergistically prevented hyperglycemia and significantly ameliorated glucose tolerance and insulin resistance in diabetic mice. Taken together, IHMT-MST1-39 is a promising anti-diabetic candidate as a single agent or in combination therapy for both T1D and T2D.

Multiple targets related to mitochondrial function unveiled by metabolomics and proteomics profiles of hearts from atrial fibrillation patients.

Background: The prominent mitochondrial metabolic changes of the atrium reportedly have significant impact on electrical signals and structural remodeling which play important roles in the occurrence and development of atrial fibrillation (AF). However, the mechanism is not completely known. Objective: This study was aimed to explore the mitochondrial metabolism reprogrammed in AF patients by integrating metabolomics as well as proteomics of human atrium tissues. Methods and Results: Left atrial tissue samples were harvested from 10 non-valvular AF patients and 10 matched samples from healthy donors for transplantation. In metabolomics analysis, 113 metabolites were upregulated and 10 metabolites were downregulated in AF, where multiple pathways related to mitochondrial energy metabolism were enriched. Correlation analysis between the differentially expressed proteins and metabolites identified several hub proteins related to mitochondrial function including Glycerol-3-phosphate dehydrogenase 2 (GPD2), Synemin (SYNM), Plectin (PLEC), with MCC score of 27, 17, 16, respectively, which have the most interactions with the dysregulated metabolites and ranked at the top in network string interactions scored by MCC method. All 330 differentially expressed proteins including 225 upregulated and 105 downregulated molecules were revealed and analyzed, which identified the downregulation of GPD2 (p = 0.02 and FC = 0.77), PLEC (p < 0.001 and FC = 0.71) and SYNM (p = 0.04 and FC = 0.76) in AF patients. Gene Set Variation Analysis (GSEA) showed mitochondrial metabolism-associated pathways including oxidative phosphorylation (NES: -1.73) and ATP biosynthetic process (NES: -2.29), were dramatically diversified in human AF. In GSVA, the expression levels of GPD2, PLEC, and SYNM were demonstrated to be associated with multiple metabolic pathways related to mitochondrial function (e.g., lipid metabolism and AMP activated protein kinase signaling) and cardiac structural and electrical remodeling (e.g., contractile fiber, ion homeostasis), which were proven vital in the development and maintenance of AF. Conclusion: In all, this study provides new insights into understanding the mechanisms of AF progression, especially the reprogramming mitochondrial metabolism, and identifies several genes related to mitochondrial function as novel targets for AF, which may be involved in the occurrence and development of AF.

Douchi Peptides Vy and Sfllr Improve Glucose Homeostasis and Gut Dysbacteriosis In High-Fat Diet-Induced Insulin Resistant Mice.

Two peptides VY (Val-Tyr) and SFLLR (Ser-Phe-Leu-Leu-Arg) were recently identified from soy-fermented douchi with hypoglycemic activity in cells. The study aimed to understand their potential effects on glucose metabolism and insulin sensitivity as well as their mechanisms of action in a high-fat diet (HFD) induced insulin resistant model. C57BL/6 mice were fed HFD for 8 weeks, followed by peptide supplementation (doses: 10 and 50 mg/kg body weight) for 8 weeks. Peptides supplementation, especially SFLLR, reduced body weight gain, insulin resistance, hyperglycemia, inflammation, liver injury, and lipid accumulation. In both muscle and liver, both peptides activated glycogen synthase (GS), the key enzyme for glycogen synthesis, and also inhibited phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6PC), two rate-limiting enzymes for gluconeogenesis, via insulin and AMPK (5' adenosine monophosphate-activated protein kinase) signalling pathways. Furthermore, VY and SFLLR supplementation reversed HFD-induced gut dysbacteriosis by decreasing the abundance of Enterococcus, Oscillibacter, and Deferribacter, and also increased the abundances of Alistipes, Lactobacillus, Faecalibaculum, Akkermansia, and Bifidobacterium (usually beneficial in the intestine). Our study revealed the potential applications of peptides VY and SFLLR as a diet-based strategy for the prevention of type 2 diabetes. This article is protected by copyright. All rights reserved.

Surfactin alleviated hyperglycaemia in mice with type 2 diabetes induced by a high-fat diet and streptozotocin

Type 2 diabetes mellitus (T2DM) is associated with liver dysfunction and intestinal dysbiosis. Bioactive peptides (BAPs) have been reported to ameliorate T2DM by preventing oxidative damage to the liver. Bacillus amyloliquefaciens fmb50 produces the lipopeptide surfactin with a wide range of biological activities. The effects of surfactin on T2DM, on the other hand, have not been studied. In the present study, 80 mg/kg body weight surfactin supplementation lowered fasting blood glucose (FBG) levels by 21.05 % and insulin resistance (IR) by 18.18 % compared with those in the T2DM group, reduced inflammation, and increased antioxidant activity in mice with T2DM induced by a high-fat diet (HFD) and streptozotocin (STZ). According to further research, surfactin administration reduced Firmicutes-to-Bacteroidetes ratios while increasing Bifidobacterium abundance by 20 times and the level of the tight junction protein Occludin by 18.38 % and ZO-1 by 66.60 %. Furthermore, surfactin also improved hepatic glucose metabolism by activating the adenosine monophosphate-activated protein kinase (AMPK) signalling pathway, increasing glycogen synthesis and glucose transporter 2 (GLUT2) protein expression while reducing glucose-6-phosphatase (G6Pase) protein expression. In addition, the increased Bifidobacterium abundance indirectly reduced the liver burden of the metabolic products indole, cresol and amine produced by saprophytic bacteria. All of these findings revealed that surfactin not only ameliorated HFD/STZ-induced gut dysbiosis and preserved intestinal barrier integrity but also enhanced hepatic glucose metabolism and detoxification function in T2DM mice. The gut microbiota appeared to be important in controlling glucose metabolism, IR, fat accumulation, inflammation and antioxidation, according to Spearman’s correlation coefficients. All data indicated that surfactin alleviated hyperglycaemia in mice with T2DM induced by HFD/STZ.

Transcriptomic and proteomic studies suggest the establishment of advanced zonation-like profiles in human-induced pluripotent stem cell-derived liver sinusoidal endothelial cells and carboxypeptidase M-positive liver progenitor cells cocultured in a fluidic microenvironment.

Hepatic zonation is a physiological feature of the liver, known to be key in the regulation of the metabolism of nutrients and xenobiotics and the biotransformation of numerous substances. However, the reproduction of this phenomenon remains challenging in vitro as only part of the processes involved in the orchestration and maintenance of zonation are fully understood. The recent advances in organ-on-chip technologies, which allow for the integration of multicellular 3D tissues in a dynamic microenvironment, could offer solutions for the reproduction of zonation within a single culture vessel. An in-depth analysis of zonation-related mechanisms observed during the coculture of human-induced pluripotent stem cell (hiPSC)-derived carboxypeptidase M-positive liver progenitor cells and hiPSC-derived liver sinusoidal endothelial cells within a microfluidic biochip was carried out. Hepatic phenotypes were confirmed in terms of albumin secretion, glycogen storage, CYP450 activity, and expression of specific endothelial markers such as PECAM1, RAB5A, and CD109. Further characterization of the patterns observed in the comparison of the transcription factor motif activities, the transcriptomic signature, and the proteomic profile expressed at the inlet and the outlet of the microfluidic biochip confirmed the presence of zonation-like phenomena within the biochips. In particular, differences related to Wnt/β-catenin, transforming growth factor-β, mammalian target of rapamycin, hypoxia-inducible factor-1, and AMP-activated protein kinase signaling, to the metabolism of lipids, and cellular remolding were observed. The present study shows the interest in combining cocultures of hiPSC-derived cellular models and microfluidic technologies for reproducing in vitro complex mechanisms such as liver zonation and further incites the use of those solutions for accurate reproduction of in vivo situations.

Intestinal Reg4 deficiency confers susceptibility to high-fat diet-induced liver steatosis by increasing intestinal fat absorption in mice

Regenerating gene family member 4 (REG4) is a novel marker for enteroendocrine cells and is selectively expressed in specialised enteroendocrine cells of the small intestine. However, the exact roles of REG4 are largely unknown. In this study we investigate the effects of REG4 on the development of dietary fat-dependent liver steatosis and the mechanisms involved. Mice with intestinal-specific Reg4 deficiency (Reg4 ΔIEC ) and Reg4-floxed alleles (Reg4 fl/fl ) were generated to investigate the effects of Reg4 on diet-induced obesity and liver steatosis. Serum levels of REG4 were also measured in children with obesity using ELISA. Reg4 ΔIEC mice fed a high-fat diet demonstrated significantly increased intestinal fat absorption and were prone to obesity and hepatic steatosis. Importantly, Reg4 ΔIEC mice exhibit enhanced activation of adenosine monophosphate-activated protein kinase (AMPK) signalling and increased protein abundance of the intestinal fat transporters, as well as enzymes involved in triglyceride synthesis and packaging at the proximal small intestine. Moreover, REG4 administration reduced fat absorption, and decreased the expression of intestinal fat absorption-related proteins in cultured intestinal cells possibly via the CaMKK2-AMPK pathway. Serum REG4 levels were markedly lower in children with obesity with advanced liver steatosis (p <0.05). Serum REG4 levels were inversely correlated with levels of liver enzymes, homeostasis model assessment of insulin resistance, low-density lipoprotein cholesterol, and triglycerides. Our findings directly link Reg4 deficiency with increased fat absorption and obesity-related liver steatosis, and suggest that REG4 may provide a potential target for prevention and treatment of liver steatosis in children. Hepatic steatosis is a key histological feature of non-alcoholic fatty liver disease, which is the leading chronic liver disease in children leading to the development of metabolic diseases; however, little is known about mechanisms induced by dietary fat. Intestinal REG4 acts as a novel enteroendocrine hormone reducing high-fat-diet-induced liver steatosis with decreasing intestinal fat absorption. REG4 may be a novel target for treatment of paediatric liver steatosis from the perspective of crosstalk between intestine and liver.

Dietary (−)-Epicatechin supplementation regulates myofiber development, fillet quality, and antioxidant status of Yellow River carp (Cyprinus carpio)

(−)-Epicatechin (EC) exhibits antioxidant effects and strengthens skeletal muscle growth and differentiation in mammals. However, there are no reports about the effects of EC in fish. This study evaluated the effects of EC on muscle antioxidant capacity, myofiber development, texture characteristics, and histomorphology were also investigated through feeding trial. 300 fish (16.27 ± 0.24 g) were fed four diets with different levels of EC (0, 100, 500, and 1000 mg/kg) for 60 days. The result showed that 1000 mg/kg EC supplementation enhanced antioxidant capacity and protein content in the muscle. Meanwhile, higher expression levels of proliferating cell nuclear antigen (pcna), myogenic differentiation antigen (myod), myogenin, myosin myosin heavy chain (myhc), and myosin light chain (mylc) were detected in the muscle of EC treatment, while the myostatin (mstnb) mRNA level was decreased in the EC 1000 group, the similar trends were observed in the protein expression levels of MyoD, Myogenin, MyLc (P < 0.05). Muscle hardness and pH value were increased significantly in EC 500 and 1000 groups (P < 0.05). Additionally, myofiber density, small-sized fiber percentage, and expression of genes related to AMP-activated protein kinase (AMPK) signaling pathway was promoted in the EC groups. These results indicated that dietary EC improved fillet quality and promoted myofiber development in Yellow River carp, which might be related to AMPK pathway.

Guilt by Association: Inflammation and Shared Genetic Risk Between Stress-Related and Immune Disorders

Guilt by Association: Inflammation and Shared Genetic Risk Between Stress-Related and Immune Disorders

Regulation of Energy Metabolism and Anti-Inflammatory Activities of Mastiha Fractions from Pistacia lentiscus L. var. chia

Pistacia lentiscus L. var. chia resin (Chios Mastiha), the first natural chewing gum, is widely used in Mediterranean cuisine and has been used in traditional medicine from ancient times. Regarding its chemical composition, Chios Mastiha is known to be rich in triterpenes. Triterpenes have a similar structure to glucocorticoids (GCs), the steroid hormones that exert strong anti-inflammatory activities and play crucial roles in the regulation of cellular metabolism. To simplify the characterization of the bioactive compounds of Mastiha resin, three different polarity fractions were isolated and were further analyzed regarding their main chemical composition and an assessment of their biological activities. The biological assessment focused on the evaluation of the potential anti-proliferative, anti-inflammatory, and apoptotic activities as well as the possible interference of the three different polarity Mastiha fractions with the glucocorticoid receptor signaling, with the aim of characterizing the biochemical mechanisms of the actions of the Mastiha fraction. Applying MTT cell viability assay, luciferase/β-galactosidase assay, and Western blot analysis showed that Chios Mastiha apolar, medium-polar, and polar fractions reduced the HEK293 cell viability in a dose-dependent manner, possibly by mitochondrial-mediated induction of apoptosis. Medium-polar and polar Mastiha fractions also suppressed the GR and NF-κΒ transcriptional activation and the p65 protein levels. These activities were accompanied by the modulation of protein levels of regulatory molecules playing a crucial role in cellular energy homeostasis, such as GR, phosphoenolpyruvate carboxykinase (PEPCK), and/or peroxisome proliferator-activated receptor alpha (PPARα), and by the induction of phosphorylation and the activation of the AMP-activated protein kinase (AMPK). The medium-polar fraction was found to be enriched in triterpenes, such as lupeol, 24Z-masticadienonic acid methyl ester, and 24Z-isomasticadienonic acid methyl ester, and it was the most active one, so we propose that triterpenes in medium-polar fraction are possibly the bioactive compounds responsible for Mastiha's regulatory actions on energy metabolism and anti-inflammatory activities via interference with GR, NF-κΒ, and AMPK signaling. This highlights its potential applications in many fields of pharmaceutical, cosmetic, and nutraceutical interest.

Effects of protocatechuic acid on antioxidant capacity, mitochondrial biogenesis and skeletal muscle fiber transformation

In skeletal muscle, the increased proportion of type I muscle fibers has the potential to improve muscle atrophy and prevent human metabolic diseases. Protocatechuic acid (PCA), as a kind of anthocyanin metabolite, has antioxidant and anti-inflammatory physiological activities. The purpose of this experiment was to use mice and C2C12 myotubes to examine if PCA can induce the transformation of muscle fiber and the mechanisms involved. We found that PCA significantly increased the expression of slow myosin heavy chain (MyHC), and markedly decreased the expression of fast MyHC in gastrocnemius muscle of mice and C2C12 myotubes. In addition, PCA also enhanced the antioxidant capacity and promoted mitochondrial biogenesis in mice. Importantly, the AMP-activated protein kinase (AMPK) signaling pathway was activated and AMPK inhibitor compound C attenuated the positive effect of PCA on myofiber conversion. To sum up, we revealed that PCA was able to promote the conversion of skeletal muscle fiber from type II to type I through the AMPK signaling pathway.

Ameliorative effects of mangiferin derivative TPX on insulin resistance via PI3K/AKT and AMPK signaling pathways in human HepG2 and HL-7702 hepatocytes

BackgroundAs a multifaceted metabolic disorder, insulin resistance is accompanied by the preceding onset of type 2 diabetes mellitus, hyperinsulinemia, metabolic dysfunction-associated fatty liver disease (MAFLD) and other metabolic syndromes. Currently, the number of existing drugs and mechanism-based strategies is limited to alleviate insulin resistance in clinics. As a natural polyphenol product derivative, 1,3,6,7-tetrapropylene acyloxy-ketone (TPX) showed a significant hypoglycemic effect in our previous studies. However, whether TPX could improve hepatic insulin sensitivity was unknown. PurposeTo explore whether insulin sensitivity can be improved by the treatment with TPX and further investigate its mechanism(s) of activity. MethodsTo mimic hyperglycemia and insulin resistance in vitro, human HepG2 and HL-7702 hepatocytes were exposed to high glucose. Cellular glucose uptake, glucose consumption, glycogen synthesis, and glucose production were quantified after TPX treatment. The effects of TPX on AMP-activated protein kinase (AMPK) phosphorylation, glucose metabolism, and insulin signal transduction were evaluated by western blotting and network pharmacology analysis. The eGFP-membrane of glucose transporter type 4 (GLUT4) lentivirus transfected cells were constructed to investigate the effects of TPX on GLUT4 mobilization. Reactive oxygen species activity in high glucose-induced insulin-resistant cells was measured by DCFH-DA to show oxidative stress. ResultsTreatment with TPX improved glycogen synthesis and inhibited gluconeogenesis by regulating GSK3β, G6Pase, and PEPCK. Furthermore, high glucose-induced inhibition of glucose consumption, glucose uptake, and GLUT4-mediated membrane translocation were reverted by TPX. Accordingly, mechanistic investigations revealed that TPX interacted with AMPK protein and activated the phosphorylation of AKT, thereby improving energy homeostasis and further ameliorating hepatic insulin resistance. Network pharmacology analysis and molecular docking further confirmed AMPK as an active target of TPX. Concordantly, the pharmacological activity of TPX was reversed by the AMPK inhibitor compound C when hepatocytes were exposed to high glucose stimulation. ConclusionIn summary, our study confirmed TPX contributions to insulin resistance improvements by targeting AMPK and PI3K/AKT to restore the insulin signaling pathway, which may be an important potential treatment strategy for insulin-resistance-related diseases, including MAFLD and diabetes.