Discovery,synthesis, and structure–activity relationships of 20S-dammar-24-en-2α,3β,12β,20-tetrol (GP) derivatives as a new class of AMPKα2β1γ1 activators

As a follow-up discovery of AMPK activators from natural products, 20S-dammar-24-en-2α,3β,12β,20-tetrol (GP, 1), a dammarane-type triterpenoid, was found to have some favorable metabolic effects on dyslipidemia in Golden Syrian hamsters, and activate AMPKα2β1γ1 by around 2.4 fold with an EC₅₀ of 5.1 μM on molecular level. In order to enhance its potency at AMPK and structure–activity relationship study, GP derivatives were designed, synthesized, and evaluated in pharmacological AMPK activation assays. Structure–activity relationship analysis showed that amine at the 24-position (groups I–IV) effectively and significantly increased the potency and efficacy. GP derivatives 12 and 17–19 exhibited better potency (EC₅₀: 0.3, 0.8, 0.8, and 1.0 μM) and efficacy (fold: 3.2, 2.7, 3.0, and 2.8) in the activation of AMPK heterotrimer α2β1γ1 than positive control (AMP, EC₅₀: 1.6 μM, fold: 3.2). Furthermore, the most potent compounds 12 and 17 obviously inhibited glucose output through increasing the phosphorylation of AMPK, without affecting mitochondrial membrane potential or producing cytotoxicity.     

Translocation of Bax in rat hepatocytes cultured with ferric nitrilotriacetate

Hepatic fibrosis occurs after many years of iron overload in liver. An effective iron deposition model induced by ferric nitrilotriacetate (FeNTA) in cultured rat hepatocytes was assumed. It has been shown that treatment of rat hepatocytes with FeNTA lead to oxidative stress and hepatocyte apoptosis. Hepatocyte apoptosis can promote liver fibrosis. The mechanisms of hepatocyte apoptosis induced by FeNTA have not yet been fully elucidated. The present study demonstrated that FeNTA-induced hepatocyte apoptosis was related to Bax translocation, cytochrome c release, and caspase-3 activation.     

Iron-mediated effect of alcohol on hepatocyte differentiation in HepaRG cells

The development of alcoholic liver diseases depends on the ability of hepatocyte to proliferate and differentiate in the case of alcohol-induced injury. Our previous work showed an inhibitory effect of alcohol on hepatocyte proliferation. However, the effect of alcohol on hepatocyte differentiation has not yet been precisely characterized. In the present study, we evaluated the effect of alcohol on hepatocyte differentiation in relationship with changes of iron metabolism in HepaRG cells. This unique bipotent human cell line can differentiate into hepatocytes and biliary epithelial cells, paralleling liver development. Results showed that alcohol reduced cell viability, total protein level and enhanced hepatic enzymes leakage in differentiated HepaRG cells. Moreover, it caused cell enlargement, decreased number of hepatocyte and expression of C/EBPα as well as bile canaliculi F-actin. Alcohol increased expression of hepatic cell-specific markers and alcohol-metabolizing enzymes (ADH2, CYP2E1). This was associated with a lipid peroxidation and an iron excess expressed by an increase in total iron content, ferritin level, iron uptake as well as an overexpression of genes involved in iron transport and storage. Alcohol-induced hepatoxicity was amplified by exogenous iron via exceeding iron overload. Taken together, our data demonstrate that in differentiated hepatocytes, alcohol reduces proliferation while increasing expression of hepatic cell-specific markers. Moreover, iron overload could be one of the underlying mechanisms of effect of alcohol on the whole differentiation process of hepatocytes.     

Linked decreases in liver kinase B1 and AMP-activated protein kinase activity modulate matrix catabolic responses to biomechanical injury in chondrocytes

Introduction

AMP-activated protein kinase (AMPK) maintains cultured chondrocyte matrix homeostasis in response to inflammatory cytokines. AMPK activity is decreased in human knee osteoarthritis (OA) chondrocytes. Liver kinase B1 (LKB1) is one of the upstream activators of AMPK. Hence, we examined the relationship between LKB1 and AMPK activity in OA and aging cartilages, and in chondrocytes subjected to inflammatory cytokine treatment and biomechanical compression injury, and performed translational studies of AMPK pharmacologic activation.

Methods

We assessed activity (phosphorylation) of LKB1 and AMPKα in mouse knee OA cartilage, in aging mouse cartilage (6 to 24 months), and in chondrocytes after mechanical injury by dynamic compression, via immunohistochemistry or western blot. We knocked down LKB1 by siRNA transfection. Nitric oxide, matrix metalloproteinase (MMP)-3, and MMP-13 release were measured by Griess reaction and ELISA, respectively.

Results

Knockdown of LKB1 attenuated chondrocyte AMPK activity, and increased nitric oxide, MMP-3 and MMP-13 release (P <0.05) in response to IL-1β and TNFα. Both LKB1 and AMPK activity were decreased in mouse knee OA and aged knee cartilage, and in bovine chondrocytes after biomechanical injury. Pretreatment of bovine chondrocytes with AMPK activators AICAR and A-769662 inhibited both AMPKα dephosphorylation and catabolic responses after biomechanical injury.

Conclusion

LKB1 is required for chondrocyte AMPK activity, thereby inhibiting matrix catabolic responses to inflammatory cytokines. Concurrent loss of LKB1 and AMPK activity in articular chondrocytes is associated with OA, aging and biomechanical injury. Conversely, pharmacologic AMPK activation attenuates catabolic responses to biomechanical injury, suggesting a potentially novel approach to inhibit OA development and progression.     

Chronic iron overload inhibits protein secretion by adult rat hepatocytes maintained in long-term primary culture

Short-term pure cultures and long-term cocultures of adult rat hepatocytes with rat liver epithelial cells, presumably derived from primitive biliary cells, were used to define in vitro models of iron overloaded hepatocytes in order to understand the molecular mechanism responsible for liver damage occurring in patients with hemochromatosis. In vitro iron overload was obtained by daily addition of ferric nitrilotriacetate to the culture medium. A concentration of 20 microM ferric salt induced hepatocyte iron overload with minimal cytotoxicity as evaluated by cell viability, morphological changes of treated cells and cytosolic enzyme leakage into the culture medium. The effects of iron overload on protein biosynthesis and secretion were studied in both short-term pure cultures and long-term cocultures of hepatocytes. The amounts of intracellular and newly synthesized proteins were never modified by the iron treatment. Furthermore, neither the relative amounts of transferrin and albumin mRNAs nor their translational products were altered by iron overload. Moreover, no change in the transferrin isomeric forms were observed in treated cells. In contrast, a prolonged exposure of cocultured hepatocytes to 20 microM ferric salt led to a significant decrease in the amount of proteins secreted in the medium. This decrease included the two major secreted proteins, namely albumin and transferrin, and probably all other secreted proteins. These results demonstrate that iron loading alters neither the total nor the liver specific protein synthesis activity of cultured hepatocytes. They suggest that chronic overload may impede the protein secretion process.     

Phosphorylation of Serine 399 in LKB1 Protein Short Form by Protein Kinase Cζ Is Required for Its Nucleocytoplasmic Transport and Consequent AMP-activated Protein Kinase (AMPK) Activation

Two splice variants of LKB1 exist: LKB1 long form (LKB1_L) and LKB1 short form (LKB1_S). In a previous study, we demonstrated that phosphorylation of Ser-428/431 (in LKB1_L) by protein kinase Cζ (PKCζ) was essential for LKB1-mediated activation of AMP-activated protein kinase (AMPK) in response to oxidants or metformin. Paradoxically, LKB1_S also activates AMPK although it lacks Ser-428/431. Thus, we hypothesized that LKB1_S contained additional phosphorylation sites important in AMPK activation. Truncation analysis and site-directed mutagenesis were used to identify putative PKCζ phosphorylation sites in LKB1_S. Substitution of Ser-399 to alanine did not alter the activity of LKB1_S, but abolished peroxynitrite- and metformin-induced activation of AMPK. Furthermore, the phosphomimetic mutation (S399D) increased the phosphorylation of AMPK and its downstream target phospho-acetyl-coenzyme A carboxylase (ACC). PKCζ-dependent phosphorylation of Ser-399 triggered nucleocytoplasmic translocation of LKB1_S in response to metformin or peroxynitrite treatment. This effect was ablated by pharmacological and genetic inhibition of PKCζ, by inhibition of CRM1 activity and by substituting Ser-399 with alanine (S399A). Overexpression of PKCζ up-regulated metformin-mediated phosphorylation of both AMPK (Thr-172) and ACC (Ser-79), but the effect was ablated in the S399A mutant. We conclude that, similar to Ser-428/431 (in LKB1_L), Ser-399 (in LKB1_S) is a PKCζ-dependent phosphorylation site essential for nucleocytoplasmic export of LKB1_S and consequent AMPK activation.     

Modulation of mercury-induced mitochondria-dependent apoptosis by glycine in hepatocytes

Mercury (Hg) is one of the universal environmental pollutants and is responsible for various organ pathophysiology including oxidative stress-induced hepatic disorders. In the present study, we aimed to explore the protective role of glycine in Hg-induced cytotoxicity and cell death in murine hepatocytes. Exposure of mercury (20 μM), in the form HgCl₂ for 1 h, significantly enhanced the ALT and ALP leakage, increased reactive oxygen species production, reduced cell viability and distorted the antioxidant status of hepatocytes. Flow cytometric analyses shows that Hg-induced apoptotic death in hepatocytes. Mechanism of this pathophysiology involves reduced mitochondrial membrane potential, variations in Bcl-2/Bad proteins, activation of caspases and cleavage of PARP protein. In addition, Hg distinctly increased NF-κB phosphorylation in association with IKKα phosphorylation and IκBα degradation. Concurrent treatment with glycine (45 mM), however, reduced Hg-induced oxidative stress, attenuated the changes in NF-κB phosphorylation and protects hepatocytes from Hg-induced apoptotic death. Hg also distinctly increased the phosphorylation of p38, JNK and ERK mitogen-activated protein kinase (MAPKs). Glycine treatment suppressed these apoptotic events, signifying its protective role in Hg-induced hepatocyte apoptosis as referred by reduction of p38, JNK and ERK MAPK signaling pathways. Results suggest that glycine can modulate Hg-induced oxidative stress and apoptosis in hepatocytes probably because of its antioxidant activity and functioning via mitochondria-dependent pathways and could be a beneficial agent in oxidative stress-mediated liver diseases.     

SIRT1 regulates hepatocyte lipid metabolism through activating AMP-activated protein kinase

Resveratrol may protect against metabolic disease through activating SIRT1 deacetylase. Because we have recently defined AMPK activation as a key mechanism for the beneficial effects of polyphenols on hepatic lipid accumulation, hyperlipidemia, and atherosclerosis in type 1 diabetic mice, we hypothesize that polyphenol-activated SIRT1 acts upstream of AMPK signaling and hepatocellular lipid metabolism. Here we show that polyphenols, including resveratrol and the synthetic polyphenol S17834, increase SIRT1 deacetylase activity, LKB1 phosphorylation at Ser(428), and AMPK activity. Polyphenols substantially prevent the impairment in phosphorylation of AMPK and its downstream target, ACC (acetyl-CoA carboxylase), elevation in expression of FAS (fatty acid synthase), and lipid accumulation in human HepG2 hepatocytes exposed to high glucose. These effects of polyphenols are largely abolished by pharmacological and genetic inhibition of SIRT1, suggesting that the stimulation of AMPK and lipid-lowering effect of polyphenols depend on SIRT1 activity. Furthermore, adenoviral overexpression of SIRT1 stimulates the basal AMPK signaling in HepG2 cells and in the mouse liver. AMPK activation by SIRT1 also protects against FAS induction and lipid accumulation caused by high glucose. Moreover, LKB1, but not CaMKKbeta, is required for activation of AMPK by polyphenols and SIRT1. These findings suggest that SIRT1 functions as a novel upstream regulator for LKB1/AMPK signaling and plays an essential role in the regulation of hepatocyte lipid metabolism. Targeting SIRT1/LKB1/AMPK signaling by polyphenols may have potential therapeutic implications for dyslipidemia and accelerated atherosclerosis in diabetes and age-related diseases.     

Sauchinone from Saururus chinensis protects vascular inflammation by heme oxygenase-1 induction in human umbilical vein endothelial cells

Sauchinone, a diastereomeric lignan isolated from the roots of Saururus chinensis (LOUR.) BAILL. (Saururaceae), is reported to exert a variety of biological activities such as hepatoprotective, anti-inflammatory actions and inhibitory effects on bone resorption. In this study, we investigated the effect of sauchinone in suppressing cell adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) expression in high glucose stimulated human umbilical vein endothelial cells (HUVEC). Sauchinone inhibited the phosphorylation and degradation of IκB-α, as well as the nuclear translocation of nuclear factor kappa B (NF-κB) p65 caused by the stimulation of high glucose. In addition, sauchinone induced heme oxygenase (HO)-1 expression through nuclear translocation of nuclear factor E2-related factor 2 in HUVEC. The effects of sauchinone on the high glucose-induced expression of VCAM-1 and ICAM-1 and nuclear translocation of NF-κB p65 were partially reversed by transfection of the cells with HO-1 siRNA. These findings suggest that sauchinone-induced HO-1 expression plays a key role in the vascular protective effects of sauchinone in HUVEC.     

Evidence against regulation of AMP-activated protein kinase and LKB1/STRAD/MO25 activity by creatine phosphate

Muscle contraction results in phosphorylation and activation of the AMP-activated protein kinase (AMPK) by an AMPK kinase (AMPKK). LKB1/STRAD/MO25 (LKB1) is the major AMPKK in skeletal muscle; however, the activity of LKB1 is not increased by muscle contraction. This finding suggests that phosphorylation of AMPK by LKB1 is regulated by allosteric mechanisms. Creatine phosphate is depleted during skeletal muscle contraction to replenish ATP. Thus the concentration of creatine phosphate is an indicator of cellular energy status. A previous report found that creatine phosphate inhibits AMPK activity. The purpose of this study was to determine whether creatine phosphate would inhibit 1) phosphorylation of AMPK by LKB1 and 2) AMPK activity after phosphorylation by LKB1. We found that creatine phosphate did not inhibit phosphorylation of either recombinant or purified rat liver AMPK by LKB1. We also found that creatine phosphate did not inhibit 1) active recombinant alpha1beta1gamma1 or alpha2beta2gamma2 AMPK, 2) AMPK immunoprecipitated from rat liver extracts by either the alpha1 or alpha2 subunit, or 3) AMPK chromatographically purified from rat liver. Inhibition of skeletal muscle AMPK by creatine phosphate was greatly reduced or eliminated with increased AMPK purity. In conclusion, these results suggest that creatine phosphate is not a direct regulator of LKB1 or AMPK activity. Creatine phosphate may indirectly modulate AMPK activity by replenishing ATP at the onset of muscle contraction.     

Sauchinone prevents IL‐1β‐induced inflammatory response in human chondrocytes

Sauchinone is one of the active lignan isolated from Saururus chinensis, which has been considered to possess various pharmacological activities, such as antitumor, hepatoprotective, antioxidant, and anti‐inflammatory effects. However, the functional roles of sauchinone in interleukin‐1 beta (IL‐1β)‐stimulated human osteoarthritis (OA) chondrocytes are still unknown. Thus, in this study, we investigated the anti‐inflammatory effects of sauchinone in IL‐1β‐stimulated chondrocytes. Our results demonstrated that sauchinone significantly attenuated NO and PGE2 production, as well as inhibited iNOS and COX‐2 expression in IL‐1β‐stimulated OA chondrocytes. In addition, sauchinone efficiently inhibited IL‐1β‐induced MMP‐3 and MMP‐13 release in human OA chondrocytes. Furthermore, sauchinone significantly attenuated the activation of NF‐κB in human OA chondrocytes. In conclusion, we showed for the first time that sauchinone inhibited inflammatory response in IL‐1β‐stimulated human chondrocytes probably through inhibiting the activation of NF‐κB signaling pathway. These data suggest that sauchinone may be a potential agent in the treatment of OA.     

Rapamycin enhances growth inhibition on urothelial carcinoma cells through LKB1 deficiency-mediated mitochondrial dysregulation

Rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, has significant potential for application in the treatment of urothelial carcinoma (URCa) of the bladder. Previous studies have shown that regulation of the AMP-activated serine/threonine protein kinase (AMPK)–mTOR signaling pathway enhances apoptosis by inducing autophagy or mitophagy in bladder cancer. Alteration of liver kinase B1 (LKB1)-AMPK signaling leads to mitochondrial dysfunction and the accumulation of autophagy-related proteins as a result of mitophagy, resulting in enhanced cell sensitivity to drug treatments. Therefore, we hypothesized that LKB1 deficiency in URCa cells could lead to increased sensitivity to rapamycin by inducing mitochondrial defect-mediated mitophagy. To test this, we established stable LKBI-knockdown URCa cells and analyzed the effects of rapamycin on their growth. Rapamycin enhanced growth inhibition and apoptosis in stable LKB1-knockdown URCa cells and in a xenograft mouse model. In spite of the stable downregulation of LKB1 expression, rapamycin induced AMPK activation in URCa cells, causing loss of the mitochondrial membrane potential, ATP depletion, and ROS accumulation, indicating an alteration of mitochondrial biogenesis. Our findings suggest that the absence of LKB1 can be targeted to induce dysregulated mitochondrial biogenesis by rapamycin treatment in the design of novel therapeutic strategies for bladder cancer.     

M1 muscarinic receptors modify oxidative stress response to acetaminophen-induced acute liver injury

The role of muscarinic receptor subtypes in modulating acute liver injury is unknown. We detected M1 muscarinic receptor (M1R) expression in human and murine hepatocytes, and investigated the consequences of M1R deficiency on acute liver injury in vivo and inhibiting M1R activation on hepatocyte injury in vitro. Age-matched wild-type (WT) and M1R-deficient (Chrm1^−/−) male mice were injected intraperitoneally with 200 mg/kg acetaminophen (APAP) and euthanized 0, 2, 4, 16, 24, and 36 h later. Biochemical and histological parameters indicated that liver injury peaked within 16 h after APAP treatment and resolved by 24 h. Compared to WT, M1R-deficient mice had reduced intrahepatic hemorrhage and hepatocyte necrosis, reflected by an attenuated rise in serum alanine aminotransferase levels. Livers of M1R-deficient mice showed reduced hepatocyte DNA fragmentation and attenuated expression of injury cytokines (Il-1α, Il-1β, Il-6, and Fasl). In all mice hepatic glutathione levels decreased after APAP injection, but they recovered more quickly in M1R-deficient mice. During the course of APAP-induced liver injury in M1R-deficient compared to WT mice, hepatic Nrf-2, Gclc, and Nqo1 expressions increased and nitrotyrosine generation decreased. APAP metabolic pathways were not altered by M1R deficiency; expression of hepatic Cyp2e1, Cyp1a2, Cyp3a11, Cyp3a13, Car, and Pxr was similar in Chrm1^−/− and WT mice. Finally, treatment of murine AML12 hepatocytes with a novel M1R antagonist, VU0255035, attenuated H₂O₂-induced oxidative stress, prevented GSH depletion, and enhanced viability. We conclude that M1R modify hepatocyte responses to oxidative stress and that targeting M1R has therapeutic potential for toxic liver injury.     

Ablation of LKB1 in the heart leads to energy deprivation and impaired cardiac function

Energy deprivation in the myocardium is associated with impaired heart function and increased morbidity. LKB1 is a kinase that is required for activation of AMP-activated protein kinase (AMPK) as well as 13 AMPK-related protein kinases. AMPK stimulates ATP production during ischemia and prevents post-ischemic dysfunction. We used the Cre–Lox system to generate mice where LKB1 was selectively knocked out in cardiomyocytes and muscle cells (LKB1-KO) to assess the role of LKB1 on cardiac function in these mice.Heart rates of LKB1-KO mice were reduced and ventricle diameter was increased. Ex vivo, cardiac function was impaired during aerobic perfusion of isolated working hearts, and recovery of function after ischemia was reduced. Although oxidative metabolism and mitochondrial function were normal, the AMP/ATP ratio was increased in LKB1-KO hearts. This was associated with a complete ablation of AMPKα2 activity, and a stimulation of signaling through the mammalian target of rapamycin. Our results establish a critical role for LKB1 for normal cardiac function under both aerobic conditions and during recovery after ischemia. Ablation of LKB1 leads to a decreased cardiac efficiency despite normal mitochondrial oxidative metabolism.     

A decrease in S-adenosyl-L-methionine potentiates arachidonic acid cytotoxicity in primary rat hepatocytes enriched in CYP2E1

Previous studies show that treatment with a polyunsaturated fatty acid, arachidonic acid (AA), or high concentrations of cycloleucine, an inhibitor of methionine adenosyltransferase (MAT), which lowers levels of S-adenosyl-L-methionine (SAM), increased toxicity in hepatocytes from pyrazole-treated rats which expressed high levels of cytochrome P450 2E1 (CYP2E1). In this study, I used concentrations of cycloleucine or AA, which by themselves do not produce any toxicity, to evaluate whether a decrease in SAM sensitizes hepatocytes to AA toxicity, especially in hepatocytes enriched in CYP2E1. Levels of SAM were lower by 50% in hepatocytes from pyrazole- compared to saline-treated rats. Cycloleucine treatment caused a 50% decline in SAM levels with both hepatocyte preparations and SAM levels were lowest in the pyrazole-treated hepatocytes. The combination of cycloleucine plus AA produced some toxicity and apoptosis in hepatocytes from saline-treated rats but increased toxicity and apoptosis was found in the hepatocytes from pyrazole-treated rats. Cytotoxicity could be prevented by incubation with SAM, the antioxidant trolox, and the mitochondrial permeability transition inhibitor trifluoperazine. The enhanced cytotoxicity could also be protected by treating rats with chlormethiazole, a specific inhibitor of CYP2E1, thus validating the role of CYP2E1. Cycloleucine plus AA treatment elevated production of reactive oxygen species (ROS) and lipid peroxidation to greater extents with the hepatocytes from pyrazole-treated rats than that from the saline-treated rats. I hypothesize that increased production of ROS by hepatocytes enriched in CYP2E1 potentiates AA-induced lipid peroxidation and toxicity when hepatoprotective levels of SAM are lowered. Such interactions, e.g. induction of CYP2E1, decline in SAM and polyunsaturated fatty acid-induced lipid peroxidation, may contribute to alcohol-induced liver injury.     

Sauchinone inhibits the proliferation,migration and invasion of breast cancer cells by suppressing Akt-CREB-MMP13 signaling pathway

Sauchinone, a lignan isolated from Saururus chinenesis, is known to exhibit anti-inflammatory and anti-oxidant effects. Recently, sauchinone has been reported to inhibit the growth of various cancer cells, but its effects on breast cancer cells remain poorly understood. In the present study, we investigated the effects of sauchinone on the growth of breast cancer cells along with the underlying molecular mechanisms. Our results show that sauchinone treatment markedly inhibited the proliferation, migration, and invasion of breast cancer cells. Sauchinone reduced the phosphorylation of Akt, ERK, and CREB increased by transforming growth factor-β (TGF-β). In particular, sauchinone treatment suppressed the expression of matrix metalloproteinase (MMP)-13 (MMP13) by regulating the Akt-CREB signaling pathway. Sauchinone was less effective in inhibiting cell migration in Mmp13-knockdown cells than in control cells, suggesting that MMP13 may be a novel target for sauchinone. Our study suggests that sauchinone inhibits the growth of breast cancer cells by attenuating the Akt-CREB-MMP13 pathway. In addition, the targeted inhibition of MMP13 by sauchinone represents a promising approach for the treatment of breast cancer.