AMPK is activated during lysosomal damage via a galectin-ubiquitin signal transduction system

ABSTRACT

Lysosomal damage activates AMPK, a regulator of macroautophagy/autophagy and metabolism, and elicits a strong ubiquitination response. Here we show that the cytosolic lectin LGALS9 detects lysosomal membrane breach by binding to lumenal glycoepitopes, and directs both the ubiquitination response and AMPK activation. Proteomic analyses have revealed increased LGALS9 association with lysosomes, and concomitant changes in LGALS9 interactions with its newly identified partners that control ubiquitination-deubiquitination processes. An LGALS9-inetractor, deubiquitinase USP9X, dissociates from damaged lysosomes upon recognition of lumenal glycans by LGALS9. USP9X’s departure from lysosomes promotes K63 ubiquitination and stimulation of MAP3K7/TAK1, an upstream kinase and activator of AMPK hitherto orphaned for a precise physiological function. Ubiquitin-activated MAP3K7/TAK1 controls AMPK specifically during lysosomal injury, caused by a spectrum of membrane-damaging or -permeabilizing agents, including silica crystals, the intracellular pathogen Mycobacterium tuberculosis, TNFSF10/TRAIL signaling, and the anti-diabetes drugs metformin. The LGALS9-ubiquitin system activating AMPK represents a novel signal transduction system contributing to various physiological outputs that are under the control of AMPK, including autophagy, MTOR, lysosomal maintenance and biogenesis, immunity, defense against microbes, and metabolic reprograming.     

AMPK in microvascular complications of diabetes and the beneficial effects of AMPK activators from plants

BackgroundDiabetes mellitus is a multifactorial disorder with the risk of micro- and macro-vascular complications. High glucose-induced derangements in metabolic pathways are primarily associated with the initiation and progression of secondary complications namely, diabetic nephropathy, neuropathy, and retinopathy. Adenosine monophosphate-activated protein kinase (AMPK) has emerged as an attractive therapeutic target to treat various metabolic disorders including diabetes mellitus. It is a master metabolic regulator that helps in maintaining cellular energy homeostasis by promoting ATP-generating catabolic pathways and inhibiting ATP-consuming anabolic pathways. Numerous pharmacological and plant-derived bioactive compounds that increase AMP-activated protein kinase activation has shown beneficial effects by mitigating secondary complications namely retinopathy, nephropathy, and neuropathy.PurposeThe purpose of this review is to highlight current knowledge on the role of AMPK and its activators from plant origin in diabetic microvascular complications.MethodsSearch engines such as Google Scholar, PubMed, Science Direct and Web of Science are used to extract papers using relevant key words. Papers mainly focusing on the role of AMPK and AMPK activators from plant origin in diabetic nephropathy, retinopathy, and neuropathy was chosen to be highlighted.ResultsAccording to results, decrease in AMPK activation during diabetes play a causative role in the pathogenesis of diabetic microvascular complications. Some of the plant-derived bioactive compounds were beneficial in restoring AMPK activity and ameliorating diabetic microvascular complications.ConclusionAMPK activators from plant origin are beneficial in mitigating diabetic microvascular complications. These pieces of evidence will be helpful in the development of AMPK-centric therapies to mitigate diabetic microvascular complications.     

The influence of the <Emphasis Type="Italic">PRKAG3</Emphasis> mutation on glycogen,enzyme activities and fibre types in different skeletal muscles of exercise trained pigs

Background  

AMP-activated protein kinase (AMPK) plays an important role in the regulation of glucose and lipid metabolism in skeletal muscle. Many pigs of Hampshire origin have a naturally occurring dominant mutation in the AMPK γ3 subunit. Pigs carrying this PRKAG3 (R225Q) mutation have, compared to non-carriers, higher muscle glycogen levels and increased oxidative capacity in m. longissimus dorsi, containing mainly type II glycolytic fibres. These metabolic changes resemble those seen when muscles adapt to an increased physical activity level. The aim was to stimulate AMPK by exercise training and study the influence of the PRKAG3 mutation on metabolic and fibre characteristics not only in m. longissimus dorsi, but also in other muscles with different functions.     

噻唑衍生物WSF-SN-10激活AMPK的机制研究

目的:在噻唑衍生物中筛选新型AMPK激活剂并探究其激活AMPK的分子机制,以期找到副作用少的II型糖尿病治疗药物。方法:用14种噻唑衍生物处理293T细胞,用Western Blot筛选能显著提高AMPK磷酸化水平的化合物;用HPLC和FACS分别检测该化合物对细胞内AMP/ATP比值和Ca~(2+)浓度的影响,探究其激活AMPK的分子机制。结果:WSF-SN-10(2-(2-(6,6-二甲基双环[3,1,1]庚-2-亚基)肼基)-4-(4-氰基苯基)噻唑)为14种噻唑衍生物中活性最强的AMPK激活剂;20μM下WSF-SN-10激活AMPK的活性最强;用20μM WSF-SN-10处理293T细胞后,细胞内的AMP/ATP比值和LKB1的磷酸化水平分别上升至空白对照的1.94和3.04倍,同时Ca~(2+)浓度无明显变化,说明WSF-SN-10通过增加细胞内的AMP/ATP比值来激活AMPK。结论:噻唑衍生物WSF-SN-10能抑制细胞内的ATP合成来间接激活AMPK,是治疗II型糖尿病和肥胖症的潜在药物。     

Rho-Kinase Inhibition Ameliorates Metabolic Disorders through Activation of AMPK Pathway in Mice

Background

Metabolic disorders, caused by excessive calorie intake and low physical activity, are important cardiovascular risk factors. Rho-kinase, an effector protein of the small GTP-binding protein RhoA, is an important cardiovascular therapeutic target and its activity is increased in patients with metabolic syndrome. We aimed to examine whether Rho-kinase inhibition improves high-fat diet (HFD)-induced metabolic disorders, and if so, to elucidate the involvement of AMP-activated kinase (AMPK), a key molecule of metabolic conditions.

Methods and Results

Mice were fed a high-fat diet, which induced metabolic phenotypes, such as obesity, hypercholesterolemia and glucose intolerance. These phenotypes are suppressed by treatment with selective Rho-kinase inhibitor, associated with increased whole body O₂ consumption and AMPK activation in the skeletal muscle and liver. Moreover, Rho-kinase inhibition increased mRNA expression of the molecules linked to fatty acid oxidation, mitochondrial energy production and glucose metabolism, all of which are known as targets of AMPK in those tissues. In systemic overexpression of dominant-negative Rho-kinase mice, body weight, serum lipid levels and glucose metabolism were improved compared with littermate control mice. Furthermore, in AMPKα2-deficient mice, the beneficial effects of fasudil, a Rho-kinase inhibitor, on body weight, hypercholesterolemia, mRNA expression of the AMPK targets and increase of whole body O₂ consumption were absent, whereas glucose metabolism was restored by fasudil to the level in wild-type mice. In cultured mouse myocytes, pharmacological and genetic inhibition of Rho-kinase increased AMPK activity through liver kinase b1 (LKB1), with up-regulation of its targets, which effects were abolished by an AMPK inhibitor, compound C.

Conclusions

These results indicate that Rho-kinase inhibition ameliorates metabolic disorders through activation of the LKB1/AMPK pathway, suggesting that Rho-kinase is also a novel therapeutic target of metabolic disorders.     

Spatial control of AMPK signaling at subcellular compartments

Abstract

AMP-activated protein kinase (AMPK) is a master regulator of energy homeostasis that functions to restore the energy balance by phosphorylating its substrates during altered metabolic conditions. AMPK activity is tightly controlled by diverse regulators including its upstream kinases LKB1 and CaMKK2. Recent studies have also identified the localization of AMPK at different intracellular compartments as another key mechanism for regulating AMPK signaling in response to specific stimuli. This review discusses the AMPK signaling associated with different subcellular compartments, including lysosomes, endoplasmic reticulum, mitochondria, Golgi apparatus, nucleus, and cell junctions. Because altered AMPK signaling is associated with various pathologic conditions including cancer, targeting AMPK signaling in different subcellular compartments may present attractive therapeutic approaches for treatment of disease.     

Knockdown of sarcolipin (SLN) impairs substrate utilization in human skeletal muscle cells

Background

Recent studies have highlighted that uncoupling of sarco-/endoplasmic reticulum Ca²⁺-ATPase (SERCA) by sarcolipin (SLN) increases ATP consumption and contributes to heat liberation. Exploiting this thermogenic mechanism in skeletal muscle may provide an attractive strategy to counteract obesity and associated metabolic disorders. In the present study, we have investigated the role of SLN on substrate metabolism in human skeletal muscle cells.

Methods and results

After generation of skeletal muscle cells with stable SLN knockdown (SLN-KD), cell viability, glucose and oleic acid (OA) metabolism, mitochondrial function, as well as gene expressions were determined. Depletion of SLN did not influence cell viability. However, glucose and OA oxidation were diminished in SLN-KD cells compared to control myotubes. Basal respiration measured by respirometry was also observed to be reduced in cells with SLN-KD. The metabolic perturbation in SLN-KD cells was reflected by reduced gene expression levels of peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) and forkhead box O1 (FOXO1). Furthermore, accumulation of OA was increased in cells with SLN-KD compared to control cells. These effects were accompanied by increased lipid formation and incorporation of OA into complex lipids. Additionally, formation of complex lipids and free fatty acid from de novo lipogenesis with acetate as substrate was enhanced in SLN-KD cells. Detection of lipid droplets using Oil red O staining also showed increased lipid accumulation in SLN-KD cells.

Conclusions

Overall, our study sheds light on the importance of SLN in maintaining metabolic homeostasis in human skeletal muscle. Findings from the current study suggest that therapeutic strategies involving SLN-mediated futile cycling of SERCA might have significant implications in the treatment of obesity and associated metabolic disorders.

Graphical abstract

     

Evaluation of the role of AMP-activated protein kinase and its downstream targets in mammalian hibernation

Mammalian hibernation requires an extensive reorganization of metabolism that typically includes a greater than 95% reduction in metabolic rate, selective inhibition of many ATP-consuming metabolic activities and a change in fuel use to a primary dependence on the oxidation of lipid reserves. We investigated whether the AMP-activated protein kinase (AMPK) could play a regulatory role in this reorganization. AMPK activity and the phosphorylation state of multiple downstream targets were assessed in five organs of thirteen-lined ground squirrels (Spermophilus tridecemlineatus) comparing euthermic animals with squirrels in deep torpor. AMPK activity was increased 3-fold in white adipose tissue from hibernating ground squirrels compared with euthermic controls, but activation was not seen in liver, skeletal muscle, brown adipose tissue or brain. Immunoblotting with phospho-specific antibodies revealed an increase in phosphorylation of eukaryotic elongation factor-2 at the inactivating Thr56 site in white adipose tissue, liver and brain of hibernators, but not in other tissues. Acetyl-CoA carboxylase phosphorylation at the inactivating Ser79 site was markedly increased in brown adipose tissue from hibernators, but no change was seen in white adipose tissue. No change was seen in the level of phosphorylation of the Ser565 AMPK site of hormone-sensitive lipase in adipose tissues of hibernating animals. In conclusion, AMPK does not appear to participate in the metabolic re-organization and/or the metabolic rate depression that occurs during ground squirrel hibernation.     

Physiological role of AMP-activated protein kinase in the heart: graded activation during exercise

AMP-activated protein kinase (AMPK) is emerging as a key signaling pathway that modulates cellular metabolic processes. In skeletal muscle, AMPK is activated during exercise. Increased myocardial substrate metabolism during exercise could be explained by AMPK activation. Although AMPK is known to be activated during myocardial ischemia, it remains uncertain whether AMPK is activated in response to the physiological increases in cardiac work associated with exercise. Therefore, we evaluated cardiac AMPK activity in rats at rest and after 10 min of treadmill running at moderate (15% grade, 16 m/min) or high (15% grade, 32 m/min) intensity. Total AMPK activity in the heart increased in proportion to exercise intensity (P < 0.05). AMPK activity associated with the alpha2-catalytic subunit increased 2.8 +/- 0.4-fold (P < 0.02 vs. rest) and 4.5 +/- 0.6-fold (P < 0.001 vs. rest) with moderate- and high-intensity exercise, respectively. AMPK activity associated with the alpha1-subunit increased to a lesser extent. Phosphorylation of the Thr172-regulatory site on AMPK alpha-catalytic subunits increased during exercise (P < 0.001). There was no increase in Akt phosphorylation during exercise. The changes in AMPK activity during exercise were associated with physiological AMPK effects (GLUT4 translocation to the sarcolemma and ACC phosphorylation). Thus cardiac AMPK activity increases progressively with exercise intensity, supporting the hypothesis that AMPK has a physiological role in the heart.     

Two triterpeniods from Cyclocarya paliurus (Batal) Iljinsk (Juglandaceae) promote glucose uptake in 3T3-L1 adipocytes: The relationship to AMPK activation

PurposeThe current study investigated the efficacy of Cyclocarya paliurus chloroform extract (CPEC) and its two specific triterpenoids (cyclocaric acid B and cyclocarioside H) on the regulation of glucose disposal and the underlying mechanisms in 3T3-L1 adipocytes.MethodsMice and adipocytes were stimulated by macrophages-derived conditioned medium (Mac-CM) to induce insulin resistance. CPEC was evaluated in mice for its ability by oral glucose tolerance test (OGTT) and insulin tolerance test (ITT). To investigate the hypoglycemic mechanisms of CPEC and its two triterpenoids, glucose uptake, AMP-activated protein kinase (AMPK) activation, inhibitor of NF-κB kinase β (IKKβ) phosphorylation and insulin signaling transduction were detected in 3T3-L1 adipocytes using 2-NBDG uptake assay and Western blot analysis.ResultsMac-CM, an inflammatory stimulus which induced the glucose and insulin intolerance, increased phosphorylation of IKKβ, reduced glucose uptake and impaired insulin sensitivity. CPEC and two triterpenoids improved glucose consumption and increased AMPK phosphorylation under basal and inflammatory conditions. Moreover, CPEC and its two triterpenoids not only enhanced glucose uptake in an insulin-independent manner, but also restored insulin-mediated protein kinase B (Akt) phosphorylation by reducing the activation of IKKβ and regulating insulin receptor substrate-1 (IRS-1) serine/tyrosine phosphorylation. These beneficial effects were attenuated by AMPK inhibitor compound C, implying that the effects may be associated with AMPK activation.ConclusionsCPEC and its two triterpenoids promoted glucose uptake in the absence of insulin, as well as ameliorated IRS-1/PI3K/Akt pathway by inhibiting inflammation. These effects were related to the regulation of AMPK activity.     

Antagonistic control of muscle cell size by AMPK and mTORC1

Nutrition and physical activity have profound effects on skeletal muscle metabolism and growth. Regulation of muscle mass depends on a thin balance between growth-promoting and growth-suppressing factors. Over the past decade, the mammalian target of rapamycin (mTOR) kinase has emerged as an essential factor for muscle growth by mediating the anabolic response to nutrients, insulin, insulin-like growth factors and resistance exercise. As opposed to the mTOR signaling pathway, the AMP-activated protein kinase (AMPK) is switched on during starvation and endurance exercise to upregulate energy-conserving processes. Recent evidence indicates that mTORC1 (mTOR Complex 1) and AMPK represent two antagonistic forces governing muscle adaption to nutrition, starvation and growth stimulation. Animal knockout models with impaired mTORC1 signaling showed decreased muscle mass correlated with increased AMPK activation. Interestingly, AMPK inhibition in p70S6K-deficient muscle cells restores cell growth and sensitivity to nutrients. Conversely, muscle cells lacking AMPK have increased mTORC1 activation with increased cell size and protein synthesis rate. We also demonstrated that the hypertrophic action of MyrAkt is enhanced in AMPK-deficient muscle, indicating that AMPK acts as a negative feedback control to restrain muscle hypertrophy. Our recent results extend this notion by showing that AMPKα1, but not AMPKα2, regulates muscle cell size through the control of mTORC1 signaling. These results reveal the diverse functions of the two catalytic isoforms of AMPK, with AMPKα1 playing a predominant role in the control of muscle cell size and AMPKα2 mediating muscle metabolic adaptation. Thus, the crosstalk between AMPK and mTORC1 signaling is a highly regulated way to control changes in muscle growth and metabolic rate imposed by external cues.     

Inhibition of the heterotetrameric K+ channel KCNQ1/KCNE1 by the AMP-activated protein kinase

Abstract

The heterotetrameric K⁺-channel KCNQ1/KCNE1 is expressed in heart, skeletal muscle, liver and several epithelia including the renal proximal tubule. In the heart, it contributes to the repolarization of cardiomyocytes. The repolarization is impaired in ischemia. Ischemia stimulates the AMP-activated protein kinase (AMPK), a serine/threonine kinase, sensing energy depletion and stimulating several cellular mechanisms to enhance energy production and to limit energy utilization. AMPK has previously been shown to downregulate the epithelial Na⁺ channel ENaC, an effect mediated by the ubiquitin ligase Nedd4-2. The present study explored whether AMPK regulates KCNQ1/KCNE1. To this end, cRNA encoding KCNQ1/KCNE1 was injected into Xenopus oocytes with and without additional injection of wild type AMPK (AMPKα1 + AMPKβ1 + AMPKγ1), of the constitutively active ^γR70QAMPK (α1β1γ1(R70Q)), of the kinase dead mutant ^αK45RAMPK (α1(K45R)β1γ1), or of the ubiquitin ligase Nedd4-2. KCNQ1/KCNE1 activity was determined in two electrode voltage clamp experiments. Moreover, KCNQ1 abundance in the cell membrane was determined by immunostaining and subsequent confocal imaging. As a result, wild type and constitutively active AMPK significantly reduced KCNQ1/KCNE1-mediated currents and reduced KCNQ1 abundance in the cell membrane. Similarly, Nedd4-2 decreased KCNQ1/KCNE1-mediated currents and KCNQ1 protein abundance in the cell membrane. Activation of AMPK in isolated perfused proximal renal tubules by AICAR (10 mM) was followed by significant depolarization. In conclusion, AMPK is a potent regulator of KCNQ1/KCNE1.     

Hypothalamic AMPK-induced autophagy increases food intake by regulating NPY and POMC expression

Hypothalamic AMP-activated protein kinase (AMPK) plays important roles in the regulation of food intake by altering the expression of orexigenic or anorexigenic neuropeptides. However, little is known about the mechanisms of this regulation. Here, we report that hypothalamic AMPK modulates the expression of NPY (neuropeptide Y), an orexigenic neuropeptide, and POMC (pro-opiomelanocortin-α), an anorexigenic neuropeptide, by regulating autophagic activity in vitro and in vivo. In hypothalamic cell lines subjected to low glucose availability such as 2-deoxy-d-glucose (2DG)-induced glucoprivation or glucose deprivation, autophagy was induced via the activation of AMPK, which regulates ULK1 and MTOR complex 1 followed by increased Npy and decreased Pomc expression. Pharmacological or genetic inhibition of autophagy diminished the effect of AMPK on neuropeptide expression in hypothalamic cell lines. Moreover, AMPK knockdown in the arcuate nucleus of the hypothalamus decreased autophagic activity and changed Npy and Pomc expression, leading to a reduction in food intake and body weight. AMPK knockdown abolished the orexigenic effects of intraperitoneal 2DG injection by decreasing autophagy and changing Npy and Pomc expression in mice fed a high-fat diet. We suggest that the induction of autophagy is a possible mechanism of AMPK-mediated regulation of neuropeptide expression and control of feeding in response to low glucose availability.     

WSF-P-1, a novel AMPK activator,promotes adiponectin multimerization in 3T3-L1 adipocytes

Adiponectin, an adipokine with insulin-sensitizing effect, is secreted from adipocytes into circulation as high, medium, and low molecular weight forms (HMW, MMW, and LMW). The HMW adiponectin oligomers possess the most potent insulin-sensitizing activity. WSF-P-1(N-methyl-1,2,3,4,5,6-hexahydro-1,1,5,5-tetramethyl-7H-2,4α-methanonaphthalen-7-amine) is derived from natural sesquiterpene longifolene by chemical modifications. We found that WSF-P-1 activates AMPK in both 3T3-L1 adipocytes and 293T cells in this study. Activation of AMPK by WSF-P-1 promotes the assembly of HMW adiponectin and increases the HMW/total ratio of adiponectin in 3T3-L1 adipocytes. We demonstrated that the Ca²⁺-dependent CaMKK signaling pathway is involved in WSF-P-1-induced AMPK activation and adiponectin multimerization. WSF-P-1 also activates GLUT1-mediated glucose uptake in 3T3-L1 adipocytes, making it a potential drug candidate for the treatment of type 2 diabetes, obesity, and other obesity-related metabolic diseases.     

Glucose deprivation reversibly down-regulates tissue plasminogen activator via proteasomal degradation in rat primary astrocytes

AimsTissue plasminogen activator (tPA) is an essential neuromodulator whose involvement in multiple functions such as synaptic plasticity, cytokine-like immune function and regulation of cell survival mandates rapid and tight tPA regulation in the brain. We investigated the possibility that a transient metabolic challenge induced by glucose deprivation may affect tPA activity in rat primary astrocytes, the main cell type responsible for metabolic regulation in the CNS.Main methodsRat primary astrocytes were incubated in serum-free DMEM without glucose. Casein zymography was used to determine tPA activity, and tPA mRNA was measured by RT-PCR. The signaling pathways regulating tPA activity were identified by Western blotting.Key findingsGlucose deprivation rapidly down-regulated the activity of tPA without affecting its mRNA level in rat primary astrocytes; this effect was mimicked by translational inhibitors. The down-regulation of tPA was accompanied by increased tPA degradation, which may be modulated by a proteasome-dependent degradation pathway. Glucose deprivation induced activation of PI3K-Akt-GSK3β, p38 and AMPK, and inhibition of these pathways using LY294002, SB203580 and compound C significantly inhibited glucose deprivation-induced tPA down-regulation, demonstrating the essential role of these pathways in tPA regulation in glucose-deprived astrocytes.SignificanceRapid and reversible regulation of tPA activity in rat primary astrocytes during metabolic crisis may minimize energy-requiring neurologic processes in stressed situations. This effect may thereby increase the opportunity to invest cellular resources in cell survival and may allow rapid re-establishment of normal cellular function after the crisis.     

PKA phosphorylates and inactivates AMPKα to promote efficient lipolysis

The mobilization of metabolic energy from adipocytes depends on a tightly regulated balance between hydrolysis and resynthesis of triacylglycerides (TAGs). Hydrolysis is stimulated by β‐adrenergic signalling to PKA that mediates phosphorylation of lipolytic enzymes, including hormone‐sensitive lipase (HSL). TAG resynthesis is associated with high‐energy consumption, which when inordinate, leads to increased AMPK activity that acts to restrain hydrolysis of TAGs by inhibiting PKA‐mediated activation of HSL. Here, we report that in primary mouse adipocytes, PKA associates with and phosphorylates AMPKα1 at Ser‐173 to impede threonine (Thr‐172) phosphorylation and thus activation of AMPKα1 by LKB1 in response to lipolytic signals. Activation of AMPKα1 by LKB1 is also blocked by PKA‐mediated phosphorylation of AMPKα1 in vitro. Functional analysis of an AMPKα1 species carrying a non‐phosphorylatable mutation at Ser‐173 revealed a critical function of this phosphorylation for efficient release of free fatty acids and glycerol in response to PKA‐activating signals. These results suggest a new mechanism of negative regulation of AMPK activity by PKA that is important for converting a lipolytic signal into an effective lipolytic response.     

A pull-down assay for 5' AMP-activated protein kinase activity using the GST-fused protein

An assay using a specific peptide (SAMS peptide) as a substrate is widely used for determination of AMP-activated protein kinases (AMPK) activity. However, it is not an efficient assay for crude AMPK preparations. In this study, we modified the assay by using the SAMS peptide fused to glutathione-S-transferase (GST-SAMS) instead of the SAMS peptide on its own. Radioactivity incorporated into GST-SAMS can be recovered easily by precipitation with glutathione-agarose. The kinetic parameters of partially purified AMPK for the GST-SAMS were as follows. The V_max was 0.26±0.012 nmol/min/mg of total proteins and K _m for GST-SAMS was 110±12 μM. The parameters for ATP were 0.40±0.016 nmol/min/mg of total proteins (V_max) and 202±21 μM (K _m ). The activity of AMPK in this system was stimulated about threefold by the AMPK activators, AMP or 5-amino-4-imidazolecarboxamide ribotide (ZMP), and inhibited by the AMPK inhibitors, adenine 9-β-d-arabinofuranoside (ara-A) and iodotubercidin. These values correlate well with those for the SAMS peptide reported previously. Thus, we succcssfully established a convenient and rapid method to measure AMPK applicable, even for crude enzyme preparations.     

运动干预对肥胖大鼠睾丸自噬和凋亡的影响

目的 探讨肥胖对大鼠生精小管结构及自噬和凋亡相关蛋白质的影响,并探讨运动对睾丸自噬和凋亡的影响及其调控机制。方法 将50只6周龄雄性SD大鼠随机分为标准饲养组（SD组,n=20）和高脂饲养组（HFD组,n=30）。HFD组喂养8周建立肥胖大鼠模型,并随机筛选出20只肥胖大鼠进行运动干预。SD组和HFD组分别随机分为标准对照组（CC组）、标准运动组（CE组）、肥胖对照组（OC组）、肥胖运动组（OE组）,每组10只。其中CE组和OE组进行8周中等强度跑台运动干预,60 min/d,5 d/周,其他两组维持原饲养条件。在最后一次运动结束48 h后,将大鼠腹腔麻醉,称重,取大鼠左右两侧睾丸、称量睾丸重量并计算睾丸指数。制作睾丸石蜡切片,利用HE染色法观察睾丸组织结构。采用蛋白质印迹法（Western blot）检测睾丸组织中p62、LC3II、LC3I、BCL-2、Bax和AMPK蛋白表达量并计算LC3II/LC3I比值,采用免疫荧光检测睾丸中LC3和BCL-2蛋白表达位置。结果 与CC组相比,OC组大鼠睾丸指数降低,生精小管直径显著降低（P<0.01）,精子细胞减少,睾丸组织中有脂滴沉...     

Glyceollin improves endoplasmic reticulum stress-induced insulin resistance through CaMKK-AMPK pathway in L6 myotubes

Glyceollin has been shown to have antidiabetic properties, although its molecular mechanism is not known. Here, we have investigated the metabolic effects of glyceollin in animal models of insulin resistance and in endoplasmic reticulum (ER) stress-responsive muscle cells. db/db mice were treated with glyceollin for 4 weeks and triglycerides, total cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) levels were measured. Glyceollin reduced serum insulin and triglycerides and increased HDL levels in db/db mice. Furthermore, glyceollin caused a significant improvement in glucose homeostasis without altering body weight and food intake in db/db mice. In muscle cells, glyceollin increased the activity of AMP-activated protein kinase (AMPK) as well as cellular glucose uptake. Fatty acid oxidation was also increased. In parallel, phosphorylation of acetyl-CoA carboxylase (ACC) at Ser-79 was increased, consistent with decreased ACC activity. An insulin-resistant state was induced by exposing cells to 5 μg/ml of tunicamycin as indicated by decreased insulin-mediated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and glucose uptake. Inhibition of insulin-mediated tyrosine phosphorylation of IRS-1 and glucose uptake under ER stress was prevented by glyceollin. Strikingly, glyceollin reduced ER stress-induced, c-Jun NH₂-terminal kinase activation and subsequently increased insulin signaling via stimulation of AMPK activity in L6 myotubes. Pharmacologic inhibition or knockdown of Ca²⁺/calmodulin-dependent protein kinase kinase blocked glyceollin-increased AMPK phosphorylation and insulin sensitivity under ER stress conditions. Taken together, these results indicate that glyceollin-mediated enhancement of insulin sensitivity under ER stress conditions is predominantly accomplished by activating AMPK, thereby having beneficial effects on hyperglycemia and insulin resistance.     

Poricoic acid A activates AMPK to attenuate fibroblast activation and abnormal extracellular matrix remodelling in renal fibrosis

BackgroundIn chronic kidney disease, although fibrosis prevention is beneficial, few interventions are available that specifically target fibrogenesis. Poricoic acid A (PAA) isolated from Poria cocos exhibits anti-fibrotic effects in the kidney, however the underlying mechanisms remain obscure.PurposeWe isolated PAA and investigated its effects and the underlying mechanisms in renal fibrosis.Study designUnilateral ureteral obstruction (UUO) and 5/6 nephrectomy (Nx) animal models and TGF-β1-induced renal fibroblasts (NRK-49F) were used to investigate the anti-fibrotic activity of PAA and its underlying mechanisms.MethodsWestern blots, qRT-PCR, immunofluorescence staining, co-immunoprecipitation and molecular docking methods were used. Knock-down and knock-in of adenosine monophosphate-activated protein kinase (AMPK) in the UUO model and cultured NRK-49F cells were employed to verify the mechanisms of action of PAA.ResultsPAA improved renal function and alleviated fibrosis by stimulating AMPK and inhibiting Smad3 specifically in Nx and UUO models. Reduced AMPK activity was associated with Smad3 induction, fibroblast activation, and the accumulation and aberrant remodelling of extracellular matrix (ECM) in human renal puncture samples and cultured NRK-49F cells. PAA stimulated AMPK activity and decreased fibrosis in a dose-dependent manner, thus showing that AMPK was essential for PAA to exert its anti-fibrotic effects. AMPK deficiency reduced the anti-fibrotic effects of PAA, while AMPK overexpression enhanced its effect.ConclusionPAA activated AMPK and further inhibited Smad3 specifically to suppress fibrosis by preventing aberrant ECM accumulation and remodelling and facilitating the deactivation of fibroblasts.