LKB1 Phosphorylation Research Articles

Nicorandil activates lkb1-ampk signaling pathway to ameliorate cardiac remodeling after myocardial ischemia/reperfusion injury by upregulating mt2

Abstract Background Cardiomyocyte apoptosis is a crucial event underlying the development of cardiac abnormalities and dysfunction after myocardial ischemia reperfusion (MI/R) injury. A better understanding of the cell signaling pathways involved in cardiac remodeling may support the development of new therapeutic strategies for the treatment of heart failure (HF) after MI/R. Nicorandil, an ATP-sensitive potassium channel opener, could improve mitochondrial damage and reduce oxidative stress，which reduce cardiomyocyte apoptosis. Metallothionein (Mt), reactive oxygen species (ROS) scavenger, which can reduce oxidative stress, also attenuated MI/R-induced autophagy and cell apoptosis. Except as a potassium channel opener, nicorandil may play a cardioprotective role by regulating metallothionein, and its specific mechanism remains to be elucidated. Objective To clarify the protective effect of nicorandil in myocardial ischemia/reperfusion injury, and elucidate the specific mechanism which nicorandil regulate the expression of metallothiosin to reduce myocardial ischemia/reperfusion injury, so as to provide new drug targets or new measures for clinical prevention and treatment of MI/R. Methods A cardiac MI/R injury model was constructed by the ligation of the left anterior descending coronary artery to investigate the underlying molecular mechanisms. The apoptosis of myocardial tissue cells was detected by TUNEL staining. The ultrastructural changes of myocardial tissue were observed by transmission electron microscopy. The expressions of Mt family, mitochondrial dynamics and Glucose metabolism-associated genes were measured by Western blotting and qPCR. The content of ATP was determined by ELISA in heart tissue. AutodockVina was used to evaluate binding energy and interaction patterns between drug candidates and their targets. The protein-protein interaction model was predicted by ZDOCK software. Results Treatment with nicorandil mitigated left ventricular enlargement, improved cardiac dysfunction,reduced myocardial infarcation area and decreased cardiomyocyte apoptosis after I/R. Nicorandil up-regulated the expression of Mt2 and decreased the expressions of Drp-1,p-Drp-1(ser616), while enhanced the expression of Mfn1/2,GLUT4,HK II and MPC II in myocardium. Knockdown of Mt2 decreased the effects of nicorandil on apoptosis and mitochondrial dysfunction after I/R. Mechanistically, nicorandil significantly upregulated the expression of Mt2, which could bound to LKB1 and cause phosphorylation of LKB1, resulting in AMPK-dependent activation. Conclusions Nicorandil significantly upregulated the expression of Mt2, which interacted with LKB1 to promote LKB1 protein phosphorylation, resulting in AMPK-dependent activation and subsequently alleviating I/R-induced mitochondrial dysfunction and energy metabolism disorder, and thereby reducing cardiomyocyte apoptosis, eventually improving I/ R-induced cardiac remodeling and dysfunction.

Troglitazone reduces intracellular Mycobacterium tuberculosis survival via macrophage autophagy through LKB1-AMPKα signaling.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb), results in significant morbidity and mortality worldwide. Host-directed therapy (HDT), including conventional drugs, is a promising anti-TB strategy that shows synergistic antibacterial effects when combined with anti-TB drugs. Here, the mycobactericidal effect of three anti-diabetic drugs was examined. Of these, only Troglitazone (Trog) enhanced the antimycobacterial effect in vitro and in vivo. This was due to Trog-mediated autophagy activation. Moreover, a knock-down experiment revealed that Trog activated autophagy and exhibited antimycobacterial activity through the LKB1-AMPK signaling pathway. Molecular docking and co-immunoprecipitation experiments demonstrated that Trog promoted LKB1 phosphorylation and activation by targeting STRADA. Finally, we found that Trog inhibited the intracellular survival of clinical isoniazid (INH)-resistant Mtb, and the combination of Trog and INH showed additive antibacterial effects against Mtb H37Rv. Taken together, anti-diabetic Trog may be repurposed as an HDT candidate and combined with first-line anti-TB drugs.

Abstract 2613: Chronic NNAL exposure, mimicking tobacco use in humans, deactivates LKB1 and promotes tumor progression in non-small cell lung cancer

Abstract Background: LKB1 loss-of-function is one key oncogenic event in lung cancer. Clinical data suggest that LKB1 loss-of-function is associated with patients’ smoking status. We recently discovered that NNAL, a major metabolite of a tobacco specific carcinogen NNK, rapidly induces LKB1 phosphorylation and its loss-of-function via the β-AR/PKA signaling pathway in an isomer-dependent manner in human lung cancer cells. Such an acute exposure, however, only resulted in moderate enhancement in lung cancer cell migration and chemoresistance. Here we characterized the effects of chronic NNAL exposure, better mimicking human tobacco use, on LKB1 and tumor progression in NSCLC in vitro and in vivo. Methods and Results: Human NSCLC cells with wild type LKB1 (H1299 and H1975) were cultured in the presence of 1 nM NNAL for 3, 6 and 12 months. LKB1 and its signaling pathways were characterized with phenotypes evaluated, including cell proliferation, migration, and chemoresistance. NNAL chronic exposure in lung cancer cells, mimicking its chronic exposure among smokers, resulted in more prominent LKB1 phosphorylation and the deactivation of its signaling, cell migration, and chemoresistance even in the absence of NNAL, indicating the long-lasting LKB1 loss-of-function. These observations were confirmed in a lung cancer xenograft model. More importantly, human lung cancer tissues revealed elevated LKB1 phosphorylation in comparison to the paired normal lung tissues. Conclusion: These results suggest that LKB1 loss-of-function in human lung cancer could be extended to its phosphorylation, which may be mediated by NNAL from tobacco smoke in an isomer-dependent manner via the β-AR/PKA signaling pathway. Citation Format: Tengfei Bian, Lingtao Jin, Chengguo Xing. Chronic NNAL exposure, mimicking tobacco use in humans, deactivates LKB1 and promotes tumor progression in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2613.

Phosphorylation of LKB1 by PDK1 Inhibits Cell Proliferation and Organ Growth by Decreased Activation of AMPK.

The master kinase LKB1 is a key regulator of se veral cellular processes, including cell proliferation, cell polarity and cellular metabolism. It phosphorylates and activates several downstream kinases, including AMP-dependent kinase, AMPK. Activation of AMPK by low energy supply and phosphorylation of LKB1 results in an inhibition of mTOR, thus decreasing energy-consuming processes, in particular translation and, thus, cell growth. LKB1 itself is a constitutively active kinase, which is regulated by posttranslational modifications and direct binding to phospholipids of the plasma membrane. Here, we report that LKB1 binds to Phosphoinositide-dependent kinase (PDK1) by a conserved binding motif. Furthermore, a PDK1-consensus motif is located within the kinase domain of LKB1 and LKB1 gets phosphorylated by PDK1 in vitro. In Drosophila, knockin of phosphorylation-deficient LKB1 results in normal survival of the flies, but an increased activation of LKB1, whereas a phospho-mimetic LKB1 variant displays decreased AMPK activation. As a functional consequence, cell growth as well as organism size is decreased in phosphorylation-deficient LKB1. Molecular dynamics simulations of PDK1-mediated LKB1 phosphorylation revealed changes in the ATP binding pocket, suggesting a conformational change upon phosphorylation, which in turn can alter LKB1's kinase activity. Thus, phosphorylation of LKB1 by PDK1 results in an inhibition of LKB1, decreased activation of AMPK and enhanced cell growth.

Jiedu Tongluo Baoshen formula enhances renal tubular epithelial cell autophagy to prevent renal fibrosis by activating SIRT1/LKB1/AMPK pathway

Renal fibrosis, an important pathological change in the development of diabetic kidney disease (DKD), urgently needs new treatment methods clinically. The Jiedu Tongluo Baoshen (JTBF) formula was created based on the theory of toxic damage to the kidney collaterals, and a variety of active ingredients in JTBF have inhibitory effects on epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM). In this study, the Ultra Performance Liquid Chromatography (UPLC) was employed to analyze the effective ingredients in the JTBF formula. After screening in the PubChem database, we identified 94 active compounds of JTBF and predicted the SIRT1 pathway as potential targets through network pharmacology. In addition, in the high fat diet (HFD)+Streptozocin (STZ)-induced DKD rat model and high glucose (HG)-induced NRK-52E cell model, JTBF treatment activates the phosphorylation of LKB1 and AMPK and enhances the autophagy activity of NRK-52E cells, thereby reducing the accumulation of EMT and ECM. These results have been confirmed in vivo and in vitro experiments. JTBF enhances the autophagy activity of renal tubular epithelial cells and inhibits the progression of DKD renal fibrosis by activating the SIRT1/LKB1/AMPK signal pathway. This study provides new insights into the molecular mechanism of JTBF to prevent and treat DKD renal fibrosis.

Mitophagy bridges DNA sensing with metabolic adaption to expand lung cancer stem-like cells.

While previous studies have identified cancer stem-like cells (CSCs) as a crucial driver for chemoresistance and tumor recurrence, the underlying mechanisms for populating the CSC pool remain unclear. Here, we identify hypermitophagy as a feature of human lung CSCs, promoting metabolic adaption via the Notch1-AMPK axis to drive CSC expansion. Specifically, mitophagy is highly active in CSCs, resulting in increased mitochondrial DNA (mtDNA) content in the lysosome. Lysosomal mtDNA acts as an endogenous ligand for Toll-like receptor 9 (TLR9) that promotes Notch1 activity. Notch1 interacts with AMPK to drive lysosomal AMPK activation by inducing metabolic stress and LKB1 phosphorylation. This TLR9-Notch1-AMPK axis supports mitochondrial metabolism to fuel CSC expansion. In patient-derived xenograft chimeras, targeting mitophagy and TLR9-dependent Notch1-AMPK pathway restricts tumor growth and CSC expansion. Taken together, mitochondrial hemostasis is interlinked with innate immune sensing and Notch1-AMPK activity to increase the CSC pool of human lung cancer.

SZC-6, a small-molecule activator of SIRT3, attenuates cardiac hypertrophy in mice.

Sirtuin3 (SIRT3), a class III histone deacetylase, is implicated in various cardiovascular diseases as a novel therapeutic target. SIRT3 has been proven to be cardioprotective in a model of Ang II-induced cardiac hypertrophy. However, a few small-molecule compounds targeting deacetylases could activate SIRT3. In this study, we generated a novel SIRT3 activator, 3-(2-bromo-4-hydroxyphenyl)-7-hydroxy-2H-chromen-2-one (SZC-6), through structural optimization of the first SIRT3 agonist C12. We demonstrated that SZC-6 directly bound to SIRT3 with Kd value of 15 μM, and increased SIRT3 deacetylation activity with EC50 value of 23.2 ± 3.3 µM. In neonatal rat cardiomyocytes (NRCMs), pretreatment with SZC-6 (10, 20, 40 µM) dose-dependently attenuated isoproterenol (ISO)-induced hypertrophic responses. Administration of SZC-6 (20, 40 and 60 mg·kg-1·d-1, s.c.) for 2 weeks starting from one week prior ISO treatment dose-dependently reversed ISO-induced impairment of diastolic and systolic cardiac function in wild-type mice, but not in SIRT3 knockdown mice. We showed that SZC-6 (10, 20, 40 µM) dose-dependently inhibited cardiac fibroblast proliferation and differentiation into myofibroblasts, which was abolished in SIRT3-knockdown mice. We further revealed that activation of SIRT3 by SZC-6 increased ATP production and rate of mitochondrial oxygen consumption, and reduced ROS, improving mitochondrial function in ISO-treated NRCMs. We also found that SZC-6 dose-dependently enhanced LKB1 phosphorylation, thereby promoting AMPK activation to inhibit Drp1-dependent mitochondrial fragmentation. Taken together, these results demonstrate that SZC-6 is a novel SIRT3 agonist with potential value in the treatment of cardiac hypertrophy partly through activation of the LKB1-AMPK pathway.

IL11 stimulates ERK/P90RSK to inhibit LKB1/AMPK and activate mTOR initiating a mesenchymal program in stromal, epithelial, and cancer cells

SummaryIL11 initiates fibroblast activation but also causes epithelial cell dysfunction. The mechanisms underlying these processes are not known. We report that IL11-stimulated ERK/P90RSK activity causes the phosphorylation of LKB1 at S325 and S428, leading to its inactivation. This inhibits AMPK and activates mTOR across cell types. In stromal cells, IL11-stimulated ERK activity inhibits LKB1/AMPK which is associated with mTOR activation, ⍺SMA expression, and myofibroblast transformation. In hepatocytes and epithelial cells, IL11/ERK activity inhibits LKB1/AMPK leading to mTOR activation, SNAI1 expression, and cell dysfunction. Across cells, IL11-induced phenotypes were inhibited by metformin stimulated AMPK activation. In mice, genetic or pharmacologic manipulation of IL11 activity revealed a critical role of IL11/ERK signaling for LKB1/AMPK inhibition and mTOR activation in fatty liver disease. These data identify the IL11/mTOR axis as a signaling commonality in stromal, epithelial, and cancer cells and reveal a shared IL11-driven mesenchymal program across cell types.

LKB1 phosphorylation and deactivation in lung cancer by NNAL, a metabolite of tobacco-specific carcinogen, in an isomer-dependent manner.

LKB1 loss of function is one key oncogenic event in lung cancer. Clinical data suggest that LKB1 loss of function is associated with patients' smoking status. The responsible ingredients and molecular mechanisms in tobacco for LKB1 loss of function, however, are not defined. In this study, we reported that NNAL, a major metabolite of a tobacco-specific carcinogen NNK, induces LKB1 phosphorylation and its loss of function via the β-AR/PKA signaling pathway in an isomer-dependent manner in human lung cancer cells. NNAL exposure also resulted in enhanced lung cancer cell migration and chemoresistance in an LKB1-dependent manner. A 120-day NNAL exposure in lung cancer cells, mimicking its chronic exposure among smokers, resulted in more prominent LKB1 phosphorylation, cell migration, and chemoresistance even in the absence of NNAL, indicating the long-lasting LKB1 loss of function although such an effect eventually disappeared after NNAL was removed for two months. These observations were confirmed in a lung cancer xenograft model. More importantly, human lung cancer tissues revealed elevated LKB1 phosphorylation in comparison to the paired normal lung tissues. These results suggest that LKB1 loss of function in human lung cancer could be extended to its phosphorylation, which may be mediated by NNAL from tobacco smoke in an isomer-dependent manner via the β-AR/PKA signaling pathway.

839-P: Pharmacological Inhibition of Mammalian INDY Ameliorates Western Diet–Induced NASH in Mice: Possible Implication of Fgf21-AMPK Signaling

Background: Recent data proposed a role of the citrate transporter INDY (I’m Not Dead Yet) in the development of obesity, insulin resistance and NAFLD suggesting its potential as a therapeutic target for metabolic-related disorders. The aim of our study is to determine if mammalian INDY (mINDY) inhibitors can be introduced as a new therapeutic option for NAFLD/NASH. Methods: Six-week-old C57Bl/6N mice were fed a western diet (WD) for 18 weeks to induce NASH. After 12 weeks of feeding, mice received mINDY inhibitor (PF-06649298, mINDYi; 100 mg/kg) or vehicle bid until end of week 18. During the treatment period, body weight and composition were monitored. ipGTT and FACS analysis of liver inflammatory cells were performed. Results: mINDYi-treated mice exhibited lower body weight, fat mass and higher lean mass compared to vehicle-treated mice. Results from ipGTT revealed improved glucose tolerance and insulin sensitivity. The latter was further confirmed through increased insulin-stimulated Akt phosphorylation in liver, gonadal WAT and BAT. Treatment with mINDYi attenuated WD-induced hepatic injury, steatosis and inflammation as shown histologically and biochemically by reduction in plasma ALT and AST, decrease in hepatic triglyceride accumulation and reduced hepatic lymphoid and myeloid immune cell populations. Interestingly, these effects were associated with a significant rise in plasma and hepatic Fgf21 together with increased hepatic phosphorylation of LKB1 and AMPK and decreased mTOR. Results from primary mouse hepatocytes revealed that mINDYi-induction of Fgf21 is partially dependent on AMPK activation. Conclusion: Our study shows for the first time that mINDYi attenuates diet-induced steatohepatitis; offering a promising novel treatment strategy for NAFLD. Further investigations are currently ongoing to elucidate the molecular mechanisms underlying mINDY inhibition and whether it is dependent on Fgf21-AMPK signaling. Disclosure N.El-agroudy: None. G.Zahn: Employee; Eternygen GmbH. C.Herrmann: None. G.Mingrone: Advisory Panel; Fractyl Health, Inc., Novo Nordisk, Consultant; ReCor Medical, Inc. T.C.Alves: None. A.L.Birkenfeld: None.

Ketone body and FGF21 coordinately regulate fasting-induced oxidative stress response in the heart.

Ketone body β-hydroxybutyrate (βOHB) and fibroblast growth factor-21 (FGF21) have been proposed to mediate systemic metabolic response to fasting. However, it remains elusive about the signaling elicited by ketone and FGF21 in the heart. Stimulation of neonatal rat cardiomyocytes with βOHB and FGF21 induced peroxisome proliferator-activated receptor α (PPARα) and PGC1α expression along with the phosphorylation of LKB1 and AMPK. βOHB and FGF21 induced transcription of peroxisome proliferator-activated receptor response element (PPRE)-containing genes through an activation of PPARα. Additionally, βOHB and FGF21 induced the expression of Nrf2, a master regulator for oxidative stress response, and catalase and Ucp2 genes. We evaluated the oxidative stress response gene expression after 24 h fast in global Fgf21-null (Fgf21−/−) mice, cardiomyocyte-specific FGF21-null (cmFgf21−/−) mice, wild-type (WT), and Fgf21fl/fl littermates. Fgf21−/− mice but not cmFgf21−/− mice had unexpectedly higher serum βOHB levels, and higher expression levels of PPARα and oxidative stress response genes than WT mice or Fgf21fl/fl littermates. Notably, expression levels of oxidative stress response genes were significantly correlated with serum βOHB and PGC1α levels in both WT and Fgf21−/− mice. These findings suggest that fasting-induced βOHB and circulating FGF21 coordinately regulate oxidative stress response gene expression in the heart.

Liquiritin Attenuates Pathological Cardiac Hypertrophy by Activating the PKA/LKB1/AMPK Pathway.

Background: Liquiritin (LQ) is one of the main flavonoids extracted from the roots of Glycyrrhiza spp., which are widely used in traditional Chinese medicine. Studies in both cellular and animal disease models have shown that LQ attenuates or prevents oxidative stress, inflammation, and apoptosis. However, the potential therapeutic effects of LQ on pressure overload-induced cardiac hypertrophy have not been so far explored. Therefore, we investigated the cardioprotective role of LQ and its underlying mechanisms in the aortic banding (AB)-induced cardiac hypertrophy mouse model. Methods and Results: Starting 3 days after AB surgery, LQ (80 mg/kg/day) was administered daily over 4 weeks. Echocardiography and pressure-volume loop analysis indicated that LQ treatment markedly improved hypertrophy-related cardiac dysfunction. Moreover, hematoxylin and eosin, picrosirius red, and TUNEL staining showed that LQ significantly inhibited cardiomyocyte hypertrophy, interstitial fibrosis, and apoptosis. Western blot assays further showed that LQ activated LKB1/AMPKα2/ACC signaling and inhibited mTORC1 phosphorylation in cardiomyocytes. Notably, LQ treatment failed to prevent cardiac dysfunction, hypertrophy, and fibrosis in AMPKα2 knockout (AMPKα2−/−) mice. However, LQ still induced LKB1 phosphorylation in AMPKα2−/− mouse hearts. In vitro experiments further demonstrated that LQ inhibited Ang II-induced hypertrophy in neonatal rat cardiomyocytes (NRCMs) by increasing cAMP levels and PKA activity. Supporting the central involvement of the cAMP/PKA/LKB1/AMPKα2 signaling pathway in the cardioprotective effects of LQ, inhibition of Ang II-induced hypertrophy and induction of LKB1 and AMPKα phosphorylation were no longer observed after inhibiting PKA activity. Conclusion: This study revealed that LQ alleviates pressure overload-induced cardiac hypertrophy in vivo and inhibits Ang II-induced cardiomyocyte hypertrophy in vitro via activating cAMP/PKA/LKB1/AMPKα2 signaling. These findings suggest that LQ might be a valuable adjunct to therapeutic approaches for treating pathological cardiac remodeling.

Ginsenoside CK ameliorates hepatic lipid accumulation via activating the LKB1/AMPK pathway in vitro and in vivo.

Nonalcoholic fatty liver disease (NAFLD) is a metabolic liver disease with a complex etiology, and is considered as one of the main causes of hepatocellular carcinoma (HCC). The incidence of NAFLD has presented an increasing trend annually as a result of disequilibrium in the dietary structure. However, no specific treatment has been approved for clinical therapy in NAFLD. Ginsenoside CK has been investigated given its various pharmacological activities, but its effects against NAFLD and the underlying mechanism are still unclear. In this study, fructose was used to simulate hepatic fatty degeneration in vivo, while palmitic acid (PA) and oleic acid (OA) were applied to induce lipid accumulation in vitro. The level of lipid accumulation in hepatic tissue and HepG2 cells was evaluated by Oil Red O staining. Detection of serum and liver biomarkers, western blotting, and real-time qPCR were conducted to assess the degree of hepatic steatosis. Our results indicated that ginsenoside CK could decrease the lipid deposition in HepG2 cells, retard the increase of body weight of fructose-fed mice, alleviate the lipid accumulation in serum and hepatic tissue and improve the hepatic inflammation and injury. Mechanically, ginsenoside CK modulated the expression of factors correlated with lipid synthesis and metabolism in vitro and in vivo via activating the phosphorylation of LKB1 and AMPK. Compound C, an inhibitor of AMPK, partially abrogated the beneficial effects of ginsenoside CK in HepG2 cells. In summary, ginsenoside CK acts as a LKB1/AMPK agonist to regulate the lipid metabolism and interfere with the progression of NAFLD.

Pyrroloquinoline Quinone Attenuates Fat Accumulation in Obese Mice Fed with a High-Fat Diet, Daphnia magna Supplied with a High Amount of Food, and 3T3-L1 Adipocytes

Obesity is a significant public health issue. Supplementation with pyrroloquinoline quinone (PQQ) may help prevent obesity by suppressing body fat accumulation. In the current study, we examined the effect of dietary PQQ on the inhibition of fat accumulation in obese mice, Daphnia magna, and adipocyte cells. PQQ significantly attenuated the total body fat and visceral fat volume in the abdominal region of mice. In Daphnia, body fat and body length increased with higher amounts of food; however, PQQ reduced body fat without affecting body growth. PQQ attenuated body fat accumulation under both high-fat and high-calorie consumption conditions. PQQ increased phosphorylation of LKB1 and AMPK, subsequently suppressing lipogenesis in adipocyte cell cultures. PQQ also promoted mitochondrial biogenesis by inducing the nuclear translocation of PGC1α in fat cells. Hence, dietary PQQ could prevent obesity by reducing body fat accumulation via suppressing lipogenesis and promoting mitochondrial biogenesis.

L-citrulline inhibits body weight gain and hepatic fat accumulation by improving lipid metabolism in a rat nonalcoholic fatty liver disease model.

BackgroundBody weight gain is a social issue all over the world. When body weight increased, hepatic fat accumulation also increased and it causes fatty liver disease. Therefore, developing a new treatment method and elucidating its mechanism is necessary. L‐citrulline (L‐Cit) is a free amino acid found mainly in watermelon. No reports regarding its effects on the improvement of hepatic steatosis and fibrogenesis are currently available. The aim of this study was to clarify the effect and the mechanism of L‐Cit on inhibition of body weight gain and hepatic fat accumulation in high‐fat and high‐cholesterol fed SHRSP5/Dmcr rats.MethodsL‐Cit or water (controls) was administered to six‐week‐old male SHRSP5/Dmcr rats by gavage for nine weeks. We recorded the level of body weight and food intake while performing the administration and sacrificed rats. After that, the blood and lipid metabolism‐related organs and tissues were collected and analyzed.ResultsL‐Cit treatment reduced body weight gain and hepatic TC and TG levels, and serum levels of AST and ALT. L‐Cit enhanced AMPK, LKB1, PKA, and hormone‐sensitive lipase (HSL) protein phosphorylation levels in the epididymal fat. L‐Cit treatment improved steatosis as revealed by HE staining of liver tissues and enhanced AMPK and LKB1 phosphorylation levels. Moreover, activation of Sirt1 was higher, while the liver fatty acid synthase (FAS) level was lower. Azan staining of liver sections revealed a reduction in fibrogenesis following L‐Cit treatment. Further, the liver levels of TGF‐β, Smad2/3, and α‐SMA, fibrogenesis‐related proteins and genes, were lower in the L‐Cit‐treated group.ConclusionsFrom the results of analysis of the epididymal fat and the liver, L‐Cit inhibits body weight gain and hepatic fat accumulation by activating lipid metabolism and promoting fatty acid β‐oxidation in SHRSP5/Dmcr rats.

Guanidino-Acetic Acid: A Scarce Substance in Biomass That Can Regulate Postmortem Meat Glycolysis of Broilers Subjected to Pre-slaughter Transportation.

The different substances in biomass can regulate the metabolism and reproduction of broilers. Guanidino-acetic acid (GAA) is a natural feed additive that showed a potential application in dietary for broilers, while its amount is scarce in biomass. The objective of the present study was to investigate the effects of dietary supplemented with GAA on muscle glycolysis of broilers subjected to pre-slaughter transportation. A total of 160 Qiandongnan Xiaoxiang chickens were randomly assigned into three treatments, including a basal control diet without GAA supplementation (80 birds) or supplemented with 600 mg/kg (40 birds) or 1,200 mg/kg (40 birds) GAA for 14 days. At the end of the experiment, the control group was equally divided into two groups, thus resulting in four groups. All birds in the four groups aforementioned were separately treated according to the following protocols: (1) no transport of birds of the control group fed with the basal diet; (2) a 3-h transport of birds of the control group fed with the basal diet; (3) a 3-h transport of birds fed with diets supplemented with 600 mg/kg GAA; and (4) a 3-h transport of birds fed with diets supplemented with 1,200 mg/kg GAA. The results demonstrated that 3-h pre-slaughter transport stress increased corticosterone contents and lowered glucose contents in plasma (P < 0.05), decreased pH24h (P < 0.05), and resulted in inferior meat quality evidenced by elevating the drip loss, cooking loss, and L∗ value (P < 0.05). Meanwhile, 3-h pre-slaughter transport stress decreased the contents of Cr and ATP in muscle (P < 0.05) and elevated the ratio of AMP:ATP and the glycolytic potential of muscle (P < 0.05). Moreover, 3-h pre-slaughter transport resulted in a significant elevation of mRNA expressions of LKB1 and AMPKα2 (P < 0.05), as well as the increase in protein abundances of LKB1 phosphorylation and AMPKα phosphorylation (P < 0.05). However, 1,200 mg/kg GAA supplementation alleviated negative parameters in plasma, improved meat quality, and ameliorated postmortem glycolysis and energy metabolism through regulating the creatine–phosphocreatine cycle and key factors of AMPK signaling. In conclusion, dietary supplementation with 1,200 mg/kg GAA contributed to improving meat quality via ameliorating muscle energy expenditure and delaying anaerobic glycolysis of broilers subjected to the 3-h pre-slaughter transport.

AMPK’nın Doğal Aktivatörleri ve Hastalıklarla İlişkisi

AMP (Adenozin monofosfat) ile aktifleştirilen protein kinaz (AMPK), enerji üreten yolları aktive edip enerji tüketen yolları inaktive ederek enerji homeostazını sağlayan bir düzenleyicidir. AMPK, serin/treonin kinaz ailesine aittir ve yapısal olarak üç alt birimden (α, β ve γ) oluşmaktadır. Biri hücresel AMP seviyelerinde artış, diğeri LKB1 (serine–threonine kinase liver kinase B1), CaMKKβ (Ca2+/calmodulin-dependent protein kinase β), TAK1 veya MLK3 ile α alt biriminin aktivasyon döngüsünde Thr172’nin fosforilasyonu yolu olmak üzere, AMPK iki mekanizma ile aktive edilebilmektedir. AMPK’nın aktivasyonu ile yağ asitleri, glikojen ve kolesterol sentezinin inhibasyonu sağlanırken; yağ asidi oksidasyonu, insüline bağımlı olmaksızın GLUT4 (glukoz taşıyıcı tip 4) translokasyonu ve otofaji işleminin aktivasyonu sağlanmaktadır. AMPK hastalıklar üzerine olan etkisini, çeşitli faktörler ile aktive olduktan sonra birçok metabolik ve fizyolojik yolağı düzenleyerek göstermektedir. AMPK aktivasyonunun arttırılmasının prediyabet, diyabet, obezite ve kanser gibi çeşitli hastalıkların tedavisinde önemli bir rol oynadığı görülmüştür. AMPK aktivitesini etkileyen pek çok etken bulunmaktadır. Sentetik ilaçlar, doğal bileşenler ve egzersiz bu aktivatörler arasında yer almaktadır. Son yıllarda yapılan araştırmalar, özellikle bu ürünler arasında AMPK’nın aktivasyonunu etkileyen doğal bileşenlere odaklanmıştır. Bu derlemenin amacı berberin, resveratrol, kurkumin, ginseng vb. gibi doğal bileşenlerin AMPK aktivasyonu ve hastalıklar üzerine etkisini incelemektir.

Beneficial Effect of Taraxacum coreanum Nakai via the Activation of LKB1-AMPK Signaling Pathway on Obesity.

Objective Liver kinase B (LKB) 1 and AMP-activated protein kinase (AMPK) are master regulators and sensors for energy homeostasis. AMPK is mainly activated via phosphorylation of LKB1 under energy stress. Here, we highlighted the antiobesity effect and underlying mechanism of Taraxacum coreanum Nakai (TCN) in connection with LKB1-AMPK signaling pathway. Methods Male C57BL/6 mice were fed on a high-fat diet (60% kcal fat; HFD) to induce obesity. Simultaneously, they received 100 or 200 mg/kg TCN orally for 5 weeks. We measured the body weight gain and liver weight along with liver histology. Moreover, the changes of factors related to lipid metabolism and β-oxidation were analyzed in the liver, together with blood parameters. Results The body weights were decreased in mice of the TCN200 group more than those of the HFD control group. Moreover, TCN supplementation lowered serum triglyceride (TG) and total cholesterol (TC) levels, whereas TCN increased HDL-cholesterol level. Liver pathological damage induced by HFD was alleviated with TCN treatment and accompanied with significant reduction in serum AST and ALT activities. In addition, TCN significantly increased the expression of p-AMPK compared with the HFD control group via the activation of LKB1/AMPK signaling pathway. Lipid synthesis gene like ACC was downregulated and factors related to β-oxidation such as carnitine palmitoyl transferase-1 (CPT-1) and uncoupling protein 2 (UCP-2) were upregulated through peroxisome proliferator-activated receptor (PPAR) α activation. Conclusion Taken together, these data suggest that TCN treatment regulates lipid metabolism via LKB1-AMPK signaling pathway and promotes β-oxidation by PPARα; hence, TCN may have potential remedy in the prevention and treatment of obesity.

14-Deoxy-11,12-Didehydroandrographolide Ameliorates Glucose Intolerance Enhancing the LKB1/AMPK[Formula: see text]/TBC1D1/GLUT4 Signaling Pathway and Inducing GLUT4 Expression in Myotubes and Skeletal Muscle of Obese Mice.

14-Deoxy-11,12-didehydroandrographolide (deAND), a bioactive component of Andrographis paniculata, has antidiabetic activity. AMP-activated protein kinase (AMPK) regulates glucose transport and ameliorates insulin resistance. The aim of the present study was to investigate whether activation of AMPK is involved in the mechanism by which deAND ameliorates insulin resistance in muscles. deAND amounts up to 40 [Formula: see text]M dose-dependently activated phosphorylation of AMPK[Formula: see text] and TBC1D1 in C2C12 myotubes. In addition, deAND significantly activated phosphorylation of LKB1 at 6 h after treatment, and this activation was maintained up to 48 h. deAND increased glucose uptake at 18 h after treatment, and this increase was time dependent up to 72 h. Compound C, an inhibitor of AMPK, suppressed deAND-induced phosphorylation of AMPK[Formula: see text] and TBC1D1 and reversed the effect on glucose uptake. In addition, the expression of GLUT4 mRNA and protein in C2C12 myotubes was up-regulated by deAND in a time-dependent manner. Promotion of GLUT4 gene transcription was verified by a pGL3-GLUT4 (837 bp) reporter assay. deAND also increased the nuclear translocation of MEF-2A and PPAR[Formula: see text]. After 16 weeks of feeding, the high-fat diet (HFD) inhibited phosphorylation of AMPK[Formula: see text] and TBC1D1 in skeletal muscle of obese C57BL/6JNarl mice, and deactivation of AMPK[Formula: see text] and TBC1D1 by the HFD was abolished by deAND supplementation. Supplementation with deAND significantly promoted membrane translocation of GLUT4 compared with the HFD group. Supplementation also significantly increased GLUT4 mRNA and protein expression in skeletal muscle compared with the HFD group. The hypoglycemic effects of deAND are likely associated with activation of the LKB1/AMPK[Formula: see text]/TBC1D1/GLUT4 signaling pathway and stimulation of MEF-2A- and PPAR[Formula: see text]-dependent GLUT4 gene expression, which account for the glucose uptake into skeletal muscle and lower blood glucose levels.

Lack of Augmenter of Liver Regeneration Disrupts Cholesterol Homeostasis of Liver in Mice by Inhibiting the AMPK Pathway.

It is well known that excessive cholesterol accumulation within hepatocytes deteriorates nonalcoholic fatty liver disease (NAFLD). Augmenter of liver regeneration (ALR) has been reported to alleviate NAFLD through anti‐apoptosis; however, whether ALR could protect liver from cholesterol‐induced NAFLD remains unclear. Mice with heterozygous deletion of Gfer (the gene for ALR, Gfer +/−) were generated, and liver steatosis was induced by either choline‐deficient ethionine‐supplemented, methionine choline–deficient diet for 4 weeks, or high‐fat diet for 16 weeks. The results showed that Gfer +/− mice developed a more severe fatty liver phenotype than Gfer +/+ mice. The livers of Gfer +/− mice exhibited a higher concentration of cholesterol and low‐density lipoprotein compared with the normal mice. Transcriptome‐based analysis predicts low‐density lipoprotein receptor (LDLR) primarily involved in the metabolic pathway. The experiments further indicate that cholesterol accumulation within hepatocytes is closely associated with enhancing the expression of LDLR and activation of sterol regulatory element binding protein 2 (SREBP2). Because adenosine monophosphate–activated protein kinase (AMPK) is a critical regulator of SREBP2 activation, we measured whether the activity of AMPK was regulated by ALR. We found that knockdown of ALR expression inhibited the phosphorylation of LKB1, an upstream activator of AMPK, followed by AMPK inactivation and SREBP2 maturation/nuclear translocation, leading to extensive cholesterol accumulation. Meanwhile, cellular oxidative stress increased as a result of ALR knockdown, indicating that ALR might also have a role in suppressing reactive oxygen species production. Conclusion: Our results confirm that ALR regulates cholesterol metabolism and alleviates hepatic steatosis probably through the LKB1‐AMPK‐SREBP2‐LDLR pathway in vivo and in vitro, providing a putative mechanism for combating fatty liver disease.