Ascofuranone prevents ER stress-induced insulin resistance via activation of AMP-activated protein kinase in L6 myotube cells

The current study presents that ascofuranone isolated from a phytopathogenic fungus, Ascochyta viciae, has antitumor activity against various transplantable tumors and a considerable hypolipidemic activity. AMP-activated protein kinase (AMPK) plays a critical role in cellular glucose and lipid homeostasis. We found that ascofuranone improves ER stress-induced insulin resistance by activating AMPK through the LKB1 pathway. In L6 myotube cells, ascofuranone treatment increased the phosphorylation of the Thr-172 residue of the AMPKα subunit and the Ser-79 subunit of acetyl-CoA carboxylase (ACC) and cellular glucose uptake. Ascofuranone-induced phosphorylation of AMPK and ACC was not increased in A549 cells lacking LKB1. Interestingly, ascofuranone treatment also improved insulin signaling impaired by ER stress in L6 myotube cells. These effects were all reversed by pretreatment with Compound C, an AMPK inhibitor or with adenoviral-mediated dominant-negative AMPKα2. Taken together, these results indicated that ascofuranone-mediated enhancement of glucose uptake and reduction of impaired insulin sensitivity in L6 cells is predominantly accomplished by activating AMPK, thereby mediating beneficial effects in type 2 diabetes and insulin resistance.     

Beneficial effects of beta-sitosterol on glucose and lipid metabolism in L6 myotube cells are mediated by AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that has been implicated as a key factor for controlling intracellular lipids and glucose metabolism. β-Sitosterol, a plant sterol known to prevent cardiovascular disease was identified from Schizonepeta tenuifolia to an AMPK activator. In L6 myotube cells, β-sitosterol significantly increased phosphorylation of the AMPKα subunit and acetyl-CoA carboxylase (ACC) with stimulating glucose uptake. In contrast, β-sitosterol treatment reduced intracellular levels of triglycerides and cholesterol in L6 cells. These effects were all reversed by pretreatment with AMPK inhibitor Compound C or LKB1 destabilizer radicicol. Similarly, β-sitosterol-induced phosphorylation of AMPK and ACC was not increased in HeLa cells lacking LKB1. These results together suggest that β-sitosterol-mediated enhancement of glucose uptake and reduction of triglycerides and cholesterol in L6 cells is predominantly accomplished by LKB1-mediated AMPK activation. Our findings further reveal a molecular mechanism underlying the beneficial effects of β-sitosterol on glucose and lipid metabolism.     

Effects of Exercise on AMPK Signaling and Downstream Components to PI3K in Rat with Type 2 Diabetes

Exercise can increase skeletal muscle sensitivity to insulin, improve insulin resistance and regulate glucose homeostasis in rat models of type 2 diabetes. However, the potential mechanism remains poorly understood. In this study, we established a male Sprague–Dawley rat model of type 2 diabetes, with insulin resistance and β cell dysfunction, which was induced by a high-fat diet and low-dose streptozotocin to replicate the pathogenesis and metabolic characteristics of type 2 diabetes in humans. We also investigated the possible mechanism by which chronic and acute exercise improves metabolism, and the phosphorylation and expression of components of AMP-activated protein kinase (AMPK) and downstream components of phosphatidylinositol 3-kinase (PI3K) signaling pathways in the soleus. As a result, blood glucose, triglyceride, total cholesterol, and free fatty acid were significantly increased, whereas insulin level progressively declined in diabetic rats. Interestingly, chronic and acute exercise reduced blood glucose, increased phosphorylation and expression of AMPKα1/2 and the isoforms AMPKα1 and AMPKα2, and decreased phosphorylation and expression of AMPK substrate, acetyl CoA carboxylase (ACC). Chronic exercise upregulated phosphorylation and expression of AMPK upstream kinase, LKB1. But acute exercise only increased LKB1 expression. In particular, exercise reversed the changes in protein kinase C (PKC)ζ/λ phosphorylation, and PKCζ phosphorylation and expression. Additionally, exercise also increased protein kinase B (PKB)/Akt1, Akt2 and GLUT4 expression, but AS160 protein expression was unchanged. Chronic exercise elevated Akt (Thr³⁰⁸) and (Ser⁴⁷³) and AS160 phosphorylation. Finally, we found that exercise increased peroxisome proliferator-activated receptor-γ coactivator 1 (PGC1) mRNA expression in the soleus of diabetic rats. These results indicate that both chronic and acute exercise influence the phosphorylation and expression of components of the AMPK and downstream to PIK3 (aPKC, Akt), and improve GLUT4 trafficking in skeletal muscle. These data help explain the mechanism how exercise regulates glucose homeostasis in diabetic rats.     

Xanthene Derivatives Increase Glucose Utilization through Activation of LKB1-Dependent AMP-Activated Protein Kinase

5′ AMP-activated protein kinase (AMPK) is a highly conserved serine-threonine kinase that regulates energy expenditure by activating catabolic metabolism and suppressing anabolic pathways to increase cellular energy levels. Therefore AMPK activators are considered to be drug targets for treatment of metabolic diseases such as diabetes mellitus. To identify novel AMPK activators, we screened xanthene derivatives. We determined that the AMPK activators 9H-xanthene-9-carboxylic acid {2,2,2-trichloro-1-[3-(3-nitro-phenyl)-thioureido]-ethyl}-amide (Xn) and 9H-xanthene-9-carboxylic acid {2,2,2-trichloro-1-[3-(3-cyano-phenyl)-thioureido]-ethyl}-amide (Xc) elevated glucose uptake in L6 myotubes by stimulating translocation of glucose transporter type 4 (GLUT4). Treatment with the chemical AMPK inhibitor compound C and infection with dominant-negative AMPKa2-virus inhibited AMPK phosphorylation and glucose uptake in myotubes induced by either Xn or Xc. Of the two major upstream kinases of AMPK, we found that Xn and Xc showed LKB1 dependency by knockdown of STK11, an ortholog of human LKB1. Single intravenous administration of Xn and Xc to high-fat diet-induced diabetic mice stimulated AMPK phosphorylation of skeletal muscle and improved glucose tolerance. Taken together, these results suggest that Xn and Xc regulate glucose homeostasis through LKB1-dependent AMPK activation and that the compounds are potential candidate drugs for the treatment of type 2 diabetes mellitus.     

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.     

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.     

Metformin attenuates ovarian cancer cell growth in an AMP‐kinase dispensable manner

Metformin, the most widely used drug for type 2 diabetes activates 59 adenosine monophosphate (AMP)‐activated protein kinase (AMPK), which regulates cellular energy metabolism. Here, we report that ovarian cell lines VOSE, A2780, CP70, C200, OV202, OVCAR3, SKOV3ip, PE01 and PE04 predominantly express ‐α₁, ‐β₁, ‐γ₁ and ‐γ₂ isoforms of AMPK subunits. Our studies show that metformin treatment (1) significantly inhibited proliferation of diverse chemo‐responsive and ‐resistant ovarian cancer cell lines (A2780, CP70, C200, OV202, OVCAR3, SKVO3ip, PE01 and PE04), (2) caused cell cycle arrest accompanied by decreased cyclin D1 and increased p21 protein expression, (3) activated AMPK in various ovarian cancer cell lines as evident from increased phosphorylation of AMPKα and its downstream substrate; acetyl co‐carboxylase (ACC) and enhanced β‐oxidation of fatty acid and (4) attenuated mTOR‐S6RP phosphorylation, inhibited protein translational and lipid biosynthetic pathways, thus implicating metformin as a growth inhibitor of ovarian cancer cells. We also show that metformin‐mediated effect on AMPK is dependent on liver kinase B1 (LKB1) as it failed to activate AMPK‐ACC pathway and cell cycle arrest in LKB1 null mouse embryo fibroblasts (mefs). This observation was further supported by using siRNA approach to down‐regulate LKB1 in ovarian cancer cells. In contrast, met formin inhibited cell proliferation in both wild‐type and AMPKα_1/2 null mefs as well as in AMPK silenced ovarian cancer cells. Collectively, these results provide evidence on the role of metformin as an anti‐proliferative therapeutic that can act through both AMPK‐dependent as well as AMPK‐independent pathways.     

Ginkgolic acid as a dual-targeting inhibitor for protein tyrosine phosphatases relevant to insulin resistance

Several protein tyrosine phosphatases (PTPs) that disrupt the insulin-signaling pathway were investigated by siRNAs to identify potential antidiabetic targets. Individual knockdown of PTPN9 and DUSP9 in 3T3-L1 preadipocytes increased AMPK phosphorylation, respectively, and furthermore, concurrent knockdown of both PTPN9 and DUSP9 synergistically increased AMPK phosphorylation. Next, 658 natural products were screened to identify dual inhibitors of both PTPN9 and DUSP9. Based on the selectivity and inhibition potency of the compounds, ginkgolic acid (GA) was selected for further study as a potential antidiabetic drug candidate. GA inhibited the enzymatic activity of PTPN9 (K_i = 53 µM) and DUSP9 (K_i = 2.5 µM) in vitro and resulted in a significant increase of glucose-uptake in differentiated C2C12 muscle cells and 3T3-L1 adipocytes. In addition, GA increased phosphorylation of AMPK in 3T3L1 adipocytes. In this study, GA as a dual targeting inhibitor of PTPN9 and DUSP9 increased glucose uptake in 3T3L1 and C2C12 cells by activating the AMPK signaling pathway. These results strongly suggest GA could be used as a therapeutic candidate for type 2 diabetes.     

Negative Regulation of AMP-activated Protein Kinase (AMPK) Activity by Macrophage Migration Inhibitory Factor (MIF) Family Members in Non-small Cell Lung Carcinomas

AMP-activated protein kinase (AMPK) is a nutrient- and metabolic stress-sensing enzyme activated by the tumor suppressor kinase, LKB1. Because macrophage migration inhibitory factor (MIF) and its functional homolog, d-dopachrome tautomerase (d-DT), have protumorigenic functions in non-small cell lung carcinomas (NSCLCs) but have AMPK-activating properties in nonmalignant cell types, we set out to investigate this apparent paradox. Our data now suggest that, in contrast to MIF and d-DTs AMPK-activating properties in nontransformed cells, MIF and d-DT act cooperatively to inhibit steady-state phosphorylation and activation of AMPK in LKB1 wild type and LKB1 mutant human NSCLC cell lines. Our data further indicate that MIF and d-DT, acting through their shared cell surface receptor, CD74, antagonize NSCLC AMPK activation by maintaining glucose uptake, ATP production, and redox balance, resulting in reduced Ca²⁺/calmodulin-dependent kinase kinase β-dependent AMPK activation. Combined, these studies indicate that MIF and d-DT cooperate to inhibit AMPK activation in an LKB1-independent manner.     

Infusion of a Lipid Emulsion Modulates AMPK and Related Proteins in Rat Liver,Muscle, and Adipose Tissues

The primary objective of this study was to investigate the impact of lipid oversupply on the AMPK pathway in skeletal muscle, liver, and adipose tissue. Male Wistar rats were infused with lipid emulsion (LE) or phosphate‐buffered saline for 5 h/day for 6 days. Muscles exposed to LE for 6 days exhibited increased AMPK and acetyl‐CoA carboxylase (ACC) phosphorylation, along with a greater association between AMPK and Ca²⁺/calmodulin‐dependent protein kinase kinase (CaMKK). No differences in muscle protein phosphatase 2C (PP2C) activity, LKB1 phosphorylation or AMPK and LKB1 association were observed. Muscle ACCβ, and adiponectin receptor 1 (AdipoR1) mRNA levels and PPARγ‐co‐activator 1α (PGC1α) protein levels were also increased in LE‐treated rats. In contrast, AMPK and ACC phosphorylation decreased and PP2C activity increased in rat livers exposed to LE. Hepatic mRNA levels of ACCα, PPARα, AdipoR1, AdipoR2, and sterol regulatory element–binding protein‐1c (SREBP1c) were also reduced after LE infusion. In adipose tissue, there was no significant alteration in AMPK or ACC phosphorylation. These results demonstrate that following lipid oversupply the AMPK pathway was enhanced in rat skeletal muscle while diminished in the liver and was unchanged in adipose tissue. CaMKK in skeletal muscle and PP2C in the liver, at least in part, appear to mediate these alterations. Alterations in AMPK pathway in the liver induced metabolic defects associated with lipid oversupply.     

Irisin ameliorates hepatic glucose/lipid metabolism and enhances cell survival in insulin-resistant human HepG2 cells through adenosine monophosphate-activated protein kinase signaling

Irisin is a newly identified myokine that promotes the browning of white adipose tissue, enhances glucose uptake in skeletal muscle and modulates hepatic metabolism. However, the signaling pathways involved in the effects on hepatic glucose and lipid metabolism have not been resolved. This study aimed to examine the role of irisin in the regulation of hepatic glucose/lipid metabolism and cell survival, and whether adenosine monophosphate-activated protein kinase (AMPK), a master metabolic regulator in the liver, is involved in irisin’s actions. Human liver-derived HepG2 cells were cultured in normal glucose-normal insulin (NGNI) or high glucose-high insulin (HGHI/insulin-resistant) condition. Hepatic glucose and lipid metabolism was evaluated by glucose output and glycogen content or triglyceride accumulation assays, respectively. Our results showed that irisin stimulated phosphorylation of AMPK and acetyl-CoA-carboxylase (ACC) via liver kinase B1 (LKB1) rather than Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ) in HepG2 cells. Irisin ameliorated hepatic insulin resistance induced by HGHI condition. Irisin reduced hepatic triglyceride content and glucose output, but increased glycogen content, with those effects reversed by dorsomorphin, an AMPK inhibitor. Furthermore, irisin also stimulated extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and promoted cell survival in an AMPK-dependent manner. In conclusion, our data indicate that irisin ameliorates dysregulation of hepatic glucose/lipid metabolism and cell death in insulin-resistant states via AMPK activation. These findings reveal a novel irisin-mediated protective mechanism in hepatic metabolism which provides a scientific basis for irisin as a potential therapeutic target for the treatment of insulin resistance and type 2 diabetes mellitus.     

Antidiabetic activities of extract from Malva verticillata seed via the activation of AMP-activated protein kinase

Stimulation of AMP-activated protein kinase (AMPK) signaling followed by increase of glucose uptake in L6 myotubes were studied with organic solvent extract of Malva verticillata (MV) seeds. Ethanol extract of M. verticillata seeds (MVE) significantly increased the phosphorylation level of AMPK, acetyl-CoA carboxylase (ACC), and glucose uptake in L6 myotube cells. The MVE was fractionated with n-hexane (MVE-H), chloroform (MVE-C), ethylacetate (MVE-E), n-butanol (MVE-B), and water (MVE-W). MVE-H (150 microgram/ml) showed the highest phosphorylating activity and increased glucose uptake by 2.3-fold. Oral administration of MVE-H (40 mg/kg) for 4 weeks to type 2 diabetic (db/db) mice reduced non-fasting and fasting blood glucose levels by 17.1% and 23.3%, respectively. Phosphorylation levels of AMPK and ACC in the soleus muscle and liver tissue of db/db mice were significantly increased by the administration of MVE-H. MVE-H was further fractionated using preparative HPLC to identify the AMPK-activating compounds. The NMR and GC-MS analyses revealed that β-sitosterol was a major effective compound in MVE-H. Phosphorylation levels of AMPK and ACC, and glucose uptake were significantly increased by the treatment of MVE-S (β-sitosterol) isolated from M. verticillata to L6 cells, and these effects were attenuated by an AMPK inhibitor (Compound C) pretreatment. These results, taken together, demonstrate that increased glucose uptake in L6 myotubes by MVE-H treatment is mainly accomplished through the activation of AMPK. Our finding suggests that the extract isolated from M. verticillata seed would be beneficial for the treatment of metabolic disease including type 2 diabetes and hyperlipidemia.     

The triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic-acid methyl ester has potent anti-diabetic effects in diet-induced diabetic mice and Lepr(db/db) mice

The triterpenoid 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic-acid (CDDO) and its methyl ester (CDDO-Me) are undergoing clinical trials in cancer and leukemia therapy. Here we report that CDDO-Me ameliorates diabetes in high fat diet-fed type 2 diabetic mice and in Lepr(db/db) mice. CDDO-Me reduces proinflammatory cytokine expression in these animals. Oral CDDO-Me administration reduces total body fat, plasma triglyceride, and free fatty acid levels. It also improves glucose tolerance and insulin tolerance tests. Its potent glucose-lowering activity results from enhanced insulin action. Hyperinsulinemic-euglycemic clamp reveals an increased glucose infusion rate required to maintain euglycemia and showed a significant increase in muscle-specific insulin-stimulated glucose uptake (71% soleus, 58% gastrocnemius) and peripheral glucose clearance as documented by a 48% increase in glucose disposal rate. CDDO-Me activates AMP-activated protein kinase (AMPK) and via LKB1 activation in muscle and liver in vivo. Treatment of isolated hepatocytes with CDDO-Me directly stimulates AMPK activity and LKB1 phosphorylation and decreases acetyl-coA carboxylase activity; it also down-regulates lipogenic gene expression, suppresses gluconeogenesis, and increases glucose uptake. Inhibition of AMPK phosphorylation using compound C and lentiviral-mediated knockdown of AMPK completely blocks the CDDO-Me-induced effect on hepatocytes as well as C(2)C(12) cells. We conclude that the triterpenoid CDDO-Me has potent anti-diabetic action in diabetic mouse models that is mediated at least in part through AMPK activation. The in vivo anti-diabetogenic effects occur at a dose substantially lower than that used for anti-leukemia therapy. We suggest that CDDO-Me holds promise as a potential anti-diabetic agent.     

The M3-muscarinic acetylcholine receptor stimulates glucose uptake in L6 skeletal muscle cells by a CaMKK–AMPK–dependent mechanism

The role of muscarinic acetylcholine receptors (mAChRs) in regulating glucose uptake in L6 skeletal muscle cells was investigated. [³H]-2-Deoxyglucose uptake was increased in differentiated L6 cells by insulin, acetylcholine, oxotremorine-M and carbachol. mAChR-mediated glucose uptake was inhibited by the AMPK inhibitor Compound C. Whole cell radioligand binding using [³H]-N-methyl scopolamine chloride identified mAChRs in differentiated but not undifferentiated L6 cells and M₃ mAChR mRNA was detected only in differentiated cells. M₃ mAChRs are Gq-coupled, and cholinergic stimulation by the mAChR agonists acetylcholine, oxotremorine-M and carbachol increased Ca²⁺ in differentiated but not undifferentiated L6 cells. This was due to muscarinic but not nicotinic activation as responses were antagonised by the muscarinic antagonist atropine but not the nicotinic antagonist tubocurarine. Western blotting showed that both carbachol and the AMPK activator AICAR increased phosphorylation of the AMPKα subunit at Thr172, with responses to carbachol blocked by Compound C and the CaMKK inhibitor STO609 but not by the PI3K inhibitor wortmannin. AICAR-stimulated AMPK phosphorylation was not sensitive to STO-609, confirming that this compound inhibits CaMKK but not the classical AMPK kinase LKB1. The TAK1 inhibitor (5Z)-7-oxozeaenol and the G_i inhibitor pertussis toxin both failed to block AMPK phosphorylation in response to carbachol. Using CHO-K1 cells stably expressing each of the mAChR subtypes (M₁–M₄), it was determined that only the M₁ and M₃ mAChRs phosphorylate AMPK, confirming a G_q-dependent mechanism. This study demonstrates that activation of M₃ mAChRs in L6 skeletal muscle cells stimulates glucose uptake via a CaMKK–AMPK-dependent mechanism, independent of the insulin-stimulated pathway.     

Endogenous Ligand for GPR120, Docosahexaenoic Acid,Exerts Benign Metabolic Effects on the Skeletal Muscles via AMP-activated Protein Kinase Pathway

Docosahexaenoic acid (DHA) is an endogenous ligand of G protein-coupled receptor 120 (GPR120). However, the mechanisms underlying DHA action are poorly understood. In this study, DHA stimulated glucose uptake in the skeletal muscles in an AMP-activated protein kinase (AMPK)-dependent manner. GPR120-mediated increase in intracellular Ca²⁺ was critical for DHA-mediated AMPK phosphorylation and glucose uptake. In addition, DHA stimulated GLUT4 translocation AMPK-dependently. Inhibition of AMPK and Ca²⁺/calmodulin-dependent protein kinase kinase blocked DHA-induced glucose uptake. DHA and GW9508, a GPR120 agonist, increased GPR120 expression. DHA-mediated glucose uptake was not observed in GPR120 knockdown conditions. DHA increased AMPK phosphorylation, glucose uptake, and intracellular Ca²⁺ concentration in primary cultured myoblasts. Taken together, these results indicated that the beneficial metabolic role of DHA was attributed to its ability to regulate glucose via the GPR120-mediated AMPK pathway in the skeletal muscles.     

Identification of chebulinic acid as a dual targeting inhibitor of protein tyrosine phosphatases relevant to insulin resistance

Natural products as antidiabetic agents have been shown to stimulate insulin signaling via the inhibition of the protein tyrosine phosphatases relevant to insulin resistance. Previously, we have identified PTPN9 and DUSP9 as potential antidiabetic targets and a multi-targeting natural product thereof. In this study, knockdown of PTPN11 increased AMPK phosphorylation in differentiated C2C12 muscle cells by 3.8 fold, indicating that PTPN11 could be an antidiabetic target. Screening of a library of 658 natural products against PTPN9, DUSP9, or PTPN11 identified chebulinic acid (CA) as a strong allosteric inhibitor with a slow cooperative binding to PTPN9 (IC₅₀ = 34 nM) and PTPN11 (IC₅₀ = 37 nM), suggesting that it would be a potential antidiabetic candidate. Furthermore, CA stimulated glucose uptake and resulted in increased AMP-activated protein kinase (AMPK) phosphorylation. Taken together, we demonstrated that CA increased glucose uptake as a dual inhibitor of PTPN9 and PTPN11 through activation of the AMPK signaling pathway. These results strongly suggest that CA could be used as a potential therapeutic candidate for the treatment of type 2 diabetes.     

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.     

Ca2+/calmodulin-dependent protein kinase kinase is involved in AMP-activated protein kinase activation by alpha-lipoic acid in C2C12 myotubes

alpha-Lipoic acid (ALA) widely exists in foods and is an antidiabetic agent. ALA stimulates glucose uptake and increases insulin sensitivity by the activation of AMP-activated protein kinase (AMPK) in skeletal muscle, but the underlying mechanism for AMPK activation is unknown. Here, we investigated the mechanism through which ALA activates AMPK in C2C12 myotubes. Incubation of C2C12 myotubes with 200 and 500 microM ALA increased the activity and phosphorylation of the AMPK alpha-subunit at Thr(172). Phosphorylation of the AMPK substrate, acetyl CoA carboxylase (ACC), at Ser(79) was also increased. No difference in ATP, AMP, and the calculated AMP-to-ATP ratio was observed among the different treatment groups. Since the upstream AMPK kinase, LKB1, requires an alteration of the AMP-to-ATP ratio to activate AMPK, this data showed that LKB1 might not be involved in the activation of AMPK induced by ALA. Treatment of ALA increased the intracellular Ca(2+) concentration measured by fura-2 fluorescent microscopy (P < 0.05), showing that ALA may activate AMPK through enhancing Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) signaling. Indeed, chelation of intracellular free Ca(2+) by loading cells with 25 microM BAPTA-AM for 30 min abolished the ALA-induced activation of AMPK and, in turn, phosphorylation of ACC at Ser(79). Furthermore, inhibition of CaMKK using its selective inhibitor, STO-609, abolished ALA-stimulated AMPK activation, with an accompanied reduction of ACC phosphorylation at Ser(79). In addition, ALA treatment increased the association of AMPK with CaMKK. To further show the role of CaMKK in AMPK activation, short interfering RNA was used to silence CaMKK, which abolished the ALA-induced AMPK activation. These data show that CaMKK is the kinase responsible for ALA-induced AMPK activation in C2C12 myotubes.