Astragalus polysaccharide stimulates glucose uptake in L6 myotubes through AMPK activation and AS160/TBC1D4 phosphorylation

Aim:

To establish the mechanism responsible for the stimulation of glucose uptake by Astragalus polysaccharide (APS), extracted from Astragalus membranaceus Bunge, in L6 myotubes in vitro.

Methods:

APS-stimulated glucose uptake in L6 myotubes was measured using the 2-deoxy-[³H]-D-glucose method. The adenine nucleotide contents in the cells were measured by HPLC. The phosphorylation of AMP-activated protein kinase (AMPK) and Akt substrate of 160 kDa (AS160) was examined using Western blot analysis. The cells transfected with 4P mutant AS160 (AS160-4P) were constructed using gene transfer approach.

Results:

Treatment of L6 myotubes with APS (100−1600 μg/mL) significantly increased glucose uptake in time- and concentration-dependent manners. The maximal glucose uptake was reached in the cells treated with APS (400 μg/mL) for 36 h. The APS-stimulated glucose uptake was significantly attenuated by pretreatment with Compound C, a selective AMPK inhibitor or in the cells overexpressing AS160-4P. Treatment of L6 myotubes with APS strongly promoted the activation of AMPK. We further demonstrated that either Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ) or liver kinase B1 (LKB1) mediated APS-induced activation of AMPK in L6 myotubes, and the increased cellular AMP: ATP ratio was also involved. Treatment of L6 myotubes with APS robustly enhanced the phosphorylation of AS160, which was significantly attenuated by pretreatment with Compound C.

Conclusion:

Our results demonstrate that APS stimulates glucose uptake in L6 myotubes through the AMP-AMPK-AS160 pathway, which may contribute to its hypoglycemic effect.     

Prenylflavonoids from fruit of Macaranga tanarius promote glucose uptake via AMPK activation in L6 myotubes

Skeletal muscle is a major tissue of glucose consumption and plays an important role in glucose homeostasis. Prenylflavonoids, a component of Macaranga tanarius fruits, have been reported to have antioxidant, antibacterial, and anticancer effects. However, the effects of these compounds on skeletal muscle glucose metabolism are unclear. Here, we isolated five prenylflavonoids from M. tanarius fruits, and investigated the mechanism of action of these compounds on skeletal muscle cells using L6 myotubes. We found that isonymphaeol B and 3′-geranyl naringenin increased glucose uptake in a dose-dependent manner. Furthermore, both isonymphaeol B and 3′-geranyl naringenin increased AMPK phosphorylation but did not affect PI3K-Akt phosphorylation. Isonymphaeol B and 3′-geranyl naringenin also increased Glut1 mRNA expression and plasma membrane GLUT1 protein levels. These results suggest that isonymphaeol B and 3′-geranyl naringenin have beneficial effects on glucose metabolism through AMPK and GLUT1 pathway. Isonymphaeol B and 3′-geranyl naringenin may be potential lead candidates for antidiabetic drug development.

Graphical abstract

     

小檗碱与芒果苷及其配伍对L6成纤维细胞葡萄糖摄取的影响及机制

<正>小檗碱与芒果苷皆为来源于植物的天然产物,具有抗糖尿病等多种药理活性。李学坚等[1-3]曾报道小檗碱与芒果苷配伍具有抗炎、抑菌、镇痛、降糖的药理活性,但二者配伍后在细胞水平的研究尚未见报道。该文以葡萄糖摄取为切入点,观察二者配伍对葡萄糖摄取的影响,并进而研究二者配伍对葡萄糖摄取信号通路的影响。     

Carnosic acid as a component of rosemary extract stimulates skeletal muscle cell glucose uptake via AMPK activation

Compounds that increase the activity of the energy sensor AMP‐activated kinase (AMPK) have the potential to regulate blood glucose levels. Although rosemary extract (RE) has been reported to activate AMPK and reduce blood glucose levels in vivo, the chemical components responsible for these effects are not known. In the present study, we measured the levels of the polyphenol carnosic acid (CA) in RE and examined the effects and the mechanism of action of CA on glucose transport system in muscle cells. High performance liquid chromatography (HPLC) was used to measure the levels of CA in RE. Parental and GLUT4myc or GLUT1myc overexpressing L6 rat myotubes were used. Glucose uptake was assessed using [³H]‐2‐deoxy‐d ‐glucose. Total and phosphorylated levels of Akt and AMPK were measured by immunoblotting. Plasma membrane GLUT4myc and GLUT1myc levels were examined using a GLUT translocation assay. Statistics included analysis of variance (ANOVA) followed by Tukey's post‐hoc test. At concentrations found in rosemary extract, CA stimulated glucose uptake in L6 myotubes. At 2.0 μmol/L CA a response (226 ± 9.62% of control, P=.001), similar to maximum insulin (201 ± 7.86% of control, P=.001) and metformin (213 ± 10.74% of control, P=.001) was seen. Akt phosphorylation was not affected by CA while AMPK and ACC phosphorylation was increased and the CA‐stimulated glucose uptake was significantly reduced by the AMPK inhibitor compound C. Plasma membrane GLUT4 or GLUT1 glucose transporter levels were not affected by CA. Our study shows increased muscle cell glucose uptake and AMPK activation by low CA concentrations, found in rosemary extract, indicating that CA may be responsible for the antihyperglycemic properties of rosemary extract seen in vivo.     

Selective cyclooxygenase-2 inhibitors stimulate glucose transport in L6 myotubes in a protein kinase Cdelta-dependent manner

Selective inhibitors of cyclooxygenase-2 (prostaglandin-endoperoxide synthase-2; COX-2) augment the rate of hexose uptake in myotubes by recruiting glucose transporter-4 (GLUT-4) to the plasma membrane in an insulin- and AMPKalpha-independent manner [Alpert E, Gruzman A, Lardi-Studler B, Cohen G, Reich R, Sasson S. Cyclooxygenase-2 (PTGS2) inhibitors augment the rate of hexose transport in L6 myotubes in an insulin- and AMPKalpha-independent manner. Diabetologia 2006;49:562-70]. We aimed at elucidating the molecular interactions that mediate this effect of COX-2 inhibitors in L6 myotubes. The effects of the inhibitors niflumic acid, nimesulide and rofecoxib on activities and phosphorylation state of key proteins in the insulin transduction pathway were determined. These inhibitors did not induce specific tyrosine phosphorylation in IRS-1, could not assemble a functional IRS-PI3K-PKB/Akt complex and did not activate GSK3alpha/beta, JNK1/2, ERK1/2, p38-MAPK or c-Cbl by site-specific phosphorylation(s). Yet, like insulin, they activated mTOR and induced downstream threonine phosphorylation in p70S6K and 4EBP1. However, rapamycin, which inhibits mTOR enzymatic activity, did not interfere with COX-2 inhibitor-induced stimulation of hexose uptake in myotube. Thus, mTOR activation was not required for COX-2 inhibitor-dependent augmentation of hexose transport in myotubes. Because PKCdelta has also been shown to activate mTOR, we asked whether COX-2 inhibitors activate mTOR by a prior activation of PKCdelta. Indeed, all three inhibitors induced tyrosine phosphorylation in PKCdelta and stimulated its kinase activity. Moreover, pharmacological inhibition of PKCdelta or the expression of a dominant-negative form of PKCdelta in myotubes completely abolished COX-2 inhibitor-dependent stimulation of hexose uptake. This study shows that selective COX-2 inhibitors activate a unique PKCdelta-dependent pathway to increase GLUT-4 abundance in the plasma membrane of myotubes and augment the rate of hexose transport.     

辛夷挥发油的GC指纹图谱

目的建立辛夷挥发油指纹图谱测定方法,为辛夷药材的质量控制提供可靠方法。方法采用水蒸气蒸馏法提取不同产地的辛夷的挥发油;用毛细管气相色谱技术测定指纹图谱。色谱条件:DB-17石英毛细管色谱柱(30 m×0.25 mm,0.25μm,美国Agilent公司);氢火焰离子化检测器;进样口温度为250℃;检测器温度为250℃;柱温以65℃为起始温度,以2℃.min-1升温至90℃,以8℃.min-1升温至145℃,以2℃.min-1升温至155℃,以10℃.min-1升温至250℃,保持5 min。载气为氮气,流速为1.0 mL.min-1,分流比为5∶1,进样量为1μL。结果建立了辛夷药材的GC指纹图谱,标定了23个共有峰。结论该方法可为更好的控制辛夷药材的内在质量提供科学依据。     

Chemistry and bioactivity of Flos Magnoliae, a Chinese herb for rhinitis and sinusitis

Flos Magnoliae (FM, Chinese name: Xin-yi) is one of the most commonly used Chinese medicinal herbs. It has a long history of clinical use for managing rhinitis, sinusitis and headache. More than 20 different FM species have been used clinically, which makes species identification and evaluation of pharmacological effects of individual chemical ingredients difficult. In this review, we have summarized the current knowledge on FM phytochemistry and its bioactivity activities. The bioactive compounds in FM include both lipid and water-soluble components. More than 90% of the essential components of FM species are terpenoids, including monoterpenes and sesquiterpenes. Lignans and neolignans including tetrahydrofurofuran, tetrahydrofuran and aryltetralin are also present in FM species. A small number of water-soluble compounds have been isolated from Magnolia flower buds, including a benzylisoquinoline alkaloid magnoflorine, an ester ethyl-E-p-hydroxyl-cinnamate and a flavonoid biondnoid. A wide range of pharmacological actions of FM have been reported, including anti-allergy, anti-inflammation and anti-microbial activity. The structure-activity relationship analysis revealed the influence of methylation at position 5 on the 3,7-dioxabicyclo-(3,3,0)-octane backbone of six lignans in antagonistic activities against platelet-activating factor. In addition, the trans stereoisomer fargesin had a much lower bioactivity than the cis stereoisomer demethoxyaschantin. Recent studies have been directed towards the isolation of other bioactive compounds. Further studies on FM may help to develop new anti-inflammatory and anti-allergic drugs.     

Protein kinase C-independent effects of protein kinase D3 in glucose transport in L6 myotubes

Protein kinase C (PKC) and protein kinase D (PKD) coordinate and regulate many fundamental cellular processes. In this study, we evaluate the role of classic and novel PKC (c/nPKC) and PKD in glucose transport in L6 myotubes. c/nPKC is either activated by short-term phorbol 12-myristate 13-acetate (PMA) treatment or down-regulated by prolonged PMA treatment at a high dose in L6 myotubes. Our results indicate that PMA treatments have little impact on basal and insulin-stimulated glucose uptake and insulin-induced Akt activation. In contrast, the PKC inhibitors Go6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c] carbazole], Go6983 [2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)maleimide], GF 109203X [bisindolylmaleimide I; 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(1H-indol-3-yl)maleimide], and Ro 31-8220 [bisindolylmaleimide IX; 2-{1-[3-(amidinothio)propyl]-1H-indol3-yl}-3-(1-methylindol-3-yl)maleimide] block basal and insulin-stimulated glucose uptake, and their inhibitory effects persist upon down-regulation of c/nPKC by PMA, implying the presence of PKC-independent effectors in mediating their inhibition of glucose uptake. Go6976, the potent cPKC inhibitor that also effectively inhibits PKD, dose-dependently blocks basal glucose uptake in L6 myotubes, whereas Go6983, the nonselective PKC inhibitor that is ineffective for PKD, has little effect on basal glucose uptake, implying the involvement of PKD in this process. Most prominently, adenoviral gene expression of a dominant-negative PKD isoform, PKD3, primarily inhibits basal glucose uptake and, to a lesser extent, insulin-stimulated glucose uptake, whereas overexpression of wild-type PKD3 significantly enhances basal glucose uptake. Moreover, expression of a PKD3-targeted siRNA significantly inhibits basal glucose uptake. Taken together, our results indicate that PKD, specifically PKD3, directly contributes to insulin-independent basal glucose uptake in L6 skeletal muscle cells.     

牛蒡子苷对L6骨骼肌细胞中腺苷酸活化蛋白激酶信号通路的影响

目的：探讨牛蒡子苷在L6骨骼肌细胞中对葡萄糖消耗和腺苷酸活化蛋白激酶（ AMP?activated protein kinase, AMPK）信号通路的调节作用。方法用含2％胎牛血清的1640培养基诱导L6骨骼肌细胞分化,直至细胞生长出肌管,分化完成后的L6骨骼肌细胞用含有不同浓度牛蒡子苷的培养基培养24 h后,采用葡萄糖氧化酶法检测细胞葡萄糖消耗,western blot法检测AMPK的亚单位AMPKα和磷酸化腺苷酸活化蛋白激酶（ p?AMP?activated protein kinase,p?AMPKα）的蛋白表达水平;以real?time PCR法检测过氧化物酶体增生物激活受体γ共激活因子1α（ peroxisome?proliferator?activated receptorγcoactiva?tor?1α,PGC?1α） mRNA表达水平。结果牛蒡子苷浓度为1000 g／L 时,可增加 L6骨骼肌细胞的葡萄糖消耗,差异显著（ P＜0．01）;与对照组相比,牛蒡子苷呈浓度依赖性的增强L6骨骼肌细胞中p?AMPKα蛋白表达水平;并以相同的趋势增强PGC?1α mRNA表达水平。结论牛蒡子苷增加L6骨骼肌细胞的葡萄糖消耗,并激活AMPK／PGC?1α信号通路,具有潜在的改善胰岛素抵抗的作用。     

不同蒸制时间对厚朴花中厚朴酚含量的影响

目的:确定厚朴花药材最佳蒸制时间,为优化厚朴花的炮制工艺提供依据。方法:采用RP-HPLC法,色谱柱为Agilent HC-C18柱(250 mm×4.6 mm,5μm),流动相为乙腈-甲醇-水(15∶65∶30),流速1.0 mL/min,柱温30℃,进样量10μL,检测波长294 nm,考察不同蒸制时间厚朴花中厚朴酚的含量。结果:不同蒸制时间厚朴花药材中厚朴酚含量存在差异,以蒸制8 min,40℃低温干燥的厚朴花中厚朴酚含量最高。结论:厚朴花的最佳炮制工艺为蒸制8 min后40℃低温干燥。     

用微波萃取-HPLC法测定不同产地辛夷药材中木兰脂素的含量

目的:采用微波萃取-HPLC法测定木兰脂素的含量,并考察4个不同产地辛夷药材中木兰脂素的含量差异。方法:采用微波萃取法,以75%乙醇为溶剂,在76℃恒温800 s萃取辛夷药材中的木兰脂素。Shim-Pack VP-ODS柱(150 mm×4.6 mm,5μm),流动相:乙腈-水(40∶60),流速:0.8 ml/min;柱温:室温,检测波长:238 nm,采用HPLC法测定木兰脂素的含量。结果:以峰面积(A)对浓度(c)作线性回归,回归方程为A=2.922 7×104c-3 617.4(r=0.999 9),在5.6~240.8μg/ml浓度范围内,木兰脂素的峰面积与浓度呈良好线性关系。该法的精密度、重现性和稳定性良好,低、中、高浓度(104.87、127.42和172.51μg/ml)的加样回收率分别为(100.39±1.58)%(、103.82±4.04)%和(103.74±0.13)%(n=3)。采用该法测得河南、河北、湖南、湖北4地所产的辛夷药材中木兰脂素含量分别为(6.59±0.39)%(、3.44±0.48)%、(2.73±0.28)%和(4.18±0.25)%(n=3)。其中河南产辛夷药材中木兰脂素含量最高。结论:本方法简便、准确、稳定、重复性好,可作为辛夷药材及相关制剂质量控制的定量方法。     

Platycodon grandiflorum modifies adipokines and the glucose uptake in high-fat diet in mice and L6 muscle cells

Objectives Obesity and diabetes have become the most common human health problems worldwide. Obesity's contribution to type 2 diabetes might be due to dysregulation of adipokines and glucose uptake. Methods In this study, we performed in‐vivo and in‐vitro studies to evaluate the effects of Platycodon grandiflorum extract (PGE) on adipokines and glucose uptake. Before study, platycodin D concentrations were analysed by HPLC in PGE prepared in water, in 50% ethanol and in 80% ethanol, and we selected the 80% ethanol extract as the PGE for this study based on the HPLC results. Key findings We found that inclusion of PGE in the high‐fat diet (HFD) markedly attenuated food intake, body weight, epididymal fat weight, adipocyte size and blood glucose levels by the oral glucose tolerance test in mice, and maintained serum levels of adiponectin, resistin, leptin, fructosamine and triglycerides. Gene expression analysis revealed that PGE up‐regulated adiponectin, and down‐regulated TNF‐α and leptin in fat tissue. In L6 muscle cells in vitro, PGE increased insulin‐stimulated glucose uptake. Conclusions We conclude that PGE may improve obesity in mice fed an HFD and glucose uptake in L6 muscle cells by modifying adipokines, and could offer clinical benefits as a supplement to treat obesity and diabetes.     

Rubiscolin-6 activates opioid receptors to enhance glucose uptake in skeletal muscle

Rubiscolin-6 is an opioid peptide derived from plant ribulose bisphosphate carboxylase/oxygenase (Rubisco). It has been demonstrated that opioid receptors could control glucose homeostasis in skeletal muscle independent of insulin action. Therefore, Rubiscolin-6 may be involved in the control of glucose metabolism. In the present study, we investigated the effect of rubiscolin-6 on glucose uptake in skeletal muscle. Rubiscolin-6-induced glucose uptake was measured using the fluorescent indicator 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxyglucose (2-NBDG) in L6 and C2C12 cell lines. The protein expressions of glucose transporter 4 (GLUT4) and AMP-activated protein kinase (AMPK) in L6 cells were observed by Western blotting. The in vivo effects of rubiscolin-6 were characterized in streptozotocin (STZ)-induced diabetic rats. Rubiscolin-6 induced a concentration-dependent increase in glucose uptake levels. The increase of phospho-AMPK (pAMPK) and GLUT4 expressions were also observed in L6 and C2C12 cells. Effects of rubiscolin-6 were blocked by opioid receptor antagonists and/or associated signals inhibitors. Moreover, Rubiscolin-6 produced a dose-dependent reduction of blood glucose and increased GLUT4 expression in STZ-induced diabetic rats. In conclusion, rubiscolin-6 increases glucose uptake, potentially via an activation of AMPK to enhance GLUT4 translocation after binding to opioid receptors in skeletal muscle.     

Cerebral glucose uptake in lindane-treated rats

The pesticide and ectoparasiticide lindane, gamma-isomer of hexachlorocyclohexane, is a powerful CNS-stimulant inducing convulsions and other signs of hyperexcitability in mammals. The present work was carried out to investigate the effect of lindane on brain regional glucose uptake at convulsant and non-convulsant doses. Local glucose uptake was measured in male Wistar rats using a modification of the 2-deoxyglucose (2-DG) method. Animals received i.p. [3H]2-DG and the amount of label in different brain structures of control and lindane-treated animals was assayed by a liquid scintillation counting of 18 dissected regions. Lindane at single convulsant dose (150 mg/kg, p.o.) increased 2-DG uptake in olfactory tubercules, hypothalamus, hippocampus, parafloculi and hypophysis. The uptake was decreased in parietal cortex, thalamus and pons-medulla. The pattern of 2-DG uptake after a single non-convulsant dose of 30 mg/kg p.o. was not so modified. After 1 week of treatment with 10 mg/kg per day p.o., increased 2-DG uptake was observed in superior colliculi while it was decreased in parietal cortex. The increase of 2-DG uptake in limbic regions observed at the convulsive dose agrees with the experimental association between poisoning signs induced by lindane and damage in the limbic system.     

Quercetin decreases inflammatory response and increases insulin action in skeletal muscle of ob/ob mice and in L6 myotubes

Inflammatory bowel diseases, primarily Crohn's disease and ulcerative colitis, are chronic inflammatory disorders of the gastrointestinal tract with unknown etiology. The majority of current therapeutic agents focus on controlling proinflammatory molecules. The neuropeptide nociceptin/orphanin FQ (N/OFQ) has been described as a potential immunomodulator for inflammatory bowel diseases. In this study, we asked whether the small molecule N/OFQ antagonist (-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB612111) would inhibit the development of dextran sodium sulfate-induced colitis in C57BL/6 mice. Inhibition of the N/OFQ receptor (NOP) by SB612111 significantly ameliorated the clinical disease course in these animals, as indicated by reduced fecal bleeding, improved recovery from diarrhea and weight loss, and a reduction in histopathological alterations. In addition, the inflammatory response in the colon was diminished, as demonstrated by reduced cytokine protein and messenger RNA expression for CXCL1/keratinocyte-derived chemokine, interferon-γ, interleukin-1β, interleukin-6, and tumor necrosis factor-α, some of which are known targets for the treatment of this devastating disease. Our results strongly support a role for the receptor-ligand pair NOP-N/OFQ in the pathogenesis of colitis. We conclude that inhibition of NOP receptors with small molecule inhibitors may constitute a novel, urgently needed approach for the treatment of inflammatory bowel diseases.     

Phenobarbital transiently stimulates uptake of 2-aminoisobutyric acid in hepatocytes

 Phenobarbital (PB) is a classical inducer of drug metabolizing enzymes and known to stimulate liver growth transiently in rodents. Previous studies have shown that regenerative liver growth after a partial hepatectomy is accompanied by the induction of the amino acid transport system A. In the present study we investigated whether amino acid transport is also increased by treatment of rats with PB. Na⁺-dependent hepatic uptake of the non-metabolizable amino acid 2-aminoisobutyric acid (AIB), which proceeds largely via transport system A, was studied in isolated hepatocytes from PB treated and untreated rats. Uptake of AIB (100 μM) was maximally induced (2.5-fold) 8 h after the beginning of PB treatment. Within 4 days, transport rates decreased to values similar to those determined in hepatocytes from untreated animals, despite the continuation of PB treatment. In contrast, induction of the PB-inducible cytochromes P450 2B1/2 was markedly increased during the entire experiment, as determined with the isoenzyme-selective substrate pentoxyresorufin. Kinetic analysis of AIB uptake revealed a “high” and a “low” affinity transport system. It is most likely that the high affinity system represents amino acid transport system A. Treatment with PB increased the V_max value but did not affect the apparent K_m value of the high affinity system. The present data suggest that the hepatic mitogen PB transiently induces amino acid transport system A. Received: 29 June 1995/Accepted: 28 September 1995     

The linkage of glucose to tiazofurin decreases in vitro uptake into rat glioma C6 cells

The aim of this study was to analyse the uptake of the synthetic nucleoside tiazofurin and glucoso-linker-tiazofurin conjugate (GLTC) into rat C6 glioma cells in vitro. Results indicated that C6 cells accumulated [3H] tiazofurin slowly with time and that accumulation was reduced by the presence of unlabelled GLTC in the medium which implies that GLTC competes with tiazofurin for transport sites. Uptake of [14C] 2 deoxy-glucose into these cells was very rapid and was not affected by the presence of unlabelled GLTC. To prove the true rate of uptake, the HPLC analysis of cellular extract was performed. After the 360 min of incubation in medium that contained 0.15 mM of tiazofurin, the sum of the concentration of tiazofurin and it's metabolite thiazole-adenine dinucleotide (TAD) in the cells was a total of approximately 4.8% of the amount added to each flask. After the same period of incubation in medium which contained 0.15 mM of GLTC, the sum of concentrations of conjugate, free tiazofurin and TAD represented less than 1/3 of the total concentration measured after the incubation with free tiazofurin and was further reduced in the presence of dipyridamole. Therefore, it can be concluded that GLTC shows some affinity for the nucleoside transporter, but the actual rate of uptake is low.