Flightless-1 inhibits ER stress-induced apoptosis in colorectal cancer cells by regulating Ca2+ homeostasis.

The endoplasmic reticulum (ER) stress response is an adaptive mechanism that is activated upon disruption of ER homeostasis and protects the cells against certain harmful environmental stimuli. However, critical and prolonged cell stress triggers cell death. In this study, we demonstrate that Flightless-1 (FliI) regulates ER stress-induced apoptosis in colon cancer cells by modulating Ca2+ homeostasis. FliI was highly expressed in both colon cell lines and colorectal cancer mouse models. In a mouse xenograft model using CT26 mouse colorectal cancer cells, tumor formation was slowed due to elevated levels of apoptosis in FliI-knockdown (FliI-KD) cells. FliI-KD cells treated with ER stress inducers, thapsigargin (TG), and tunicamycin exhibited activation of the unfolded protein response (UPR) and induction of UPR-related gene expression, which eventually triggered apoptosis. FliI-KD increased the intracellular Ca2+ concentration, and this upregulation was caused by accelerated ER-to-cytosolic efflux of Ca2+. The increase in intracellular Ca2+ concentration was significantly blocked by dantrolene and tetracaine, inhibitors of ryanodine receptors (RyRs). Dantrolene inhibited TG-induced ER stress and decreased the rate of apoptosis in FliI-KD CT26 cells. Finally, we found that knockdown of FliI decreased the levels of sorcin and ER Ca2+ and that TG-induced ER stress was recovered by overexpression of sorcin in FliI-KD cells. Taken together, these results suggest that FliI regulates sorcin expression, which modulates Ca2+ homeostasis in the ER through RyRs. Our findings reveal a novel mechanism by which FliI influences Ca2+ homeostasis and cell survival during ER stress.

Hovering and forward flight of the hawkmoth Manduca sexta: trim search and 6-DOF dynamic stability characterization.

We show that the forward flight speed affects the stability characteristics of the longitudinal and lateral dynamics of a flying hawkmoth; dynamic modal structures of both the planes of motion are altered due to variations in the stability derivatives. The forward flight speed u e is changed from 0.00 to 1.00 m s(-1) with an increment of 0.25 m s(-1). (The equivalent advance ratio is 0.00 to 0.38; the advance ratio is the ratio of the forward flight speed to the average wing tip speed.) As the flight speed increases, for the longitudinal dynamics, an unstable oscillatory mode becomes more unstable. Also, we show that the up/down (w(b)) dynamics become more significant at a faster flight speed due to the prominent increase in the stability derivative Z(u) (up/down force due to the forward/backward velocity). For the lateral dynamics, the decrease in the stability derivative L(v) (roll moment due to side slip velocity) at a faster flight speed affects a slightly damped stable oscillatory mode, causing it to become more stable; however, the t(half) (the time taken to reach half the amplitude) of this slightly damped stable oscillatory mode remains relatively long (∼12T at u(e) = 1 m s(-1); T is wingbeat period) compared to the other modes of motion, meaning that this mode represents the most vulnerable dynamics among the lateral dynamics at all flight speeds. To obtain the stability derivatives, trim conditions for linearization are numerically searched to find the exact trim trajectory and wing kinematics using an algorithm that uses the gradient information of a control effectiveness matrix and fully coupled six-degrees of freedom nonlinear multibody equations of motion. With this algorithm, trim conditions that consider the coupling between the dynamics and aerodynamics can be obtained. The body and wing morphology, and the wing kinematics used in this study are based on actual measurement data from the relevant literature. The aerodynamic model of the flapping wings of a hawkmoth is based on the blade element theory, and the necessary aerodynamic coefficients, including the lift, drag and wing pitching moment, are experimentally obtained from the results of previous work by the authors.

Novel phosphorylation of PPARγ ameliorates obesity-induced adipose tissue inflammation and improves insulin sensitivity.

Chronic inflammation in adipose tissue is highly associated with insulin resistance. Herein, we demonstrate that a novel modification of PPARγ is strongly associated with inflammatory responses in adipose tissue. c-Src kinase directly phosphorylated PPARγ at Tyr78, and this process was reversed by protein tyrosine phosphatase-1B (PTP-1B). In adipocytes, phosphorylation of PPARγ suppressed the expression of pro-inflammatory genes as well as the secretion of chemokines and cytokines, thus reducing macrophage migration. Importantly, pharmacological inhibition of c-Src kinase aggravated insulin resistance in obese mice with a concomitant increase in the expression of pro-inflammatory genes in adipose tissue. These data strongly suggest that PPARγ phosphorylation is the key regulatory mechanism of the inflammatory response in adipose tissue, which is highly associated with glucose tolerance and insulin sensitivity. Furthermore, these data increase our understanding of the mechanical aspects of developing novel anti-diabetic drugs targeting PPARγ phosphorylation.

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 Ca(2+) was critical for DHA-mediated AMPK phosphorylation and glucose uptake. In addition, DHA stimulated GLUT4 translocation AMPK-dependently. Inhibition of AMPK and Ca(2+)/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 Ca(2+) 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.

An improved quasi-steady aerodynamic model for insect wings that considers movement of the center of pressure.

A quasi-steady aerodynamic model in consideration of the center of pressure (C.P.) was developed for insect flight. A dynamically scaled-up robotic hawkmoth wing was used to obtain the translational lift, drag, moment and rotational force coefficients. The translational force coefficients were curve-fitted with respect to the angles of attack such that two coefficients in the Polhamus leading-edge suction analogy model were obtained. The rotational force coefficient was also compared to that derived by the standard Kutta-Joukowski theory. In order to build the accurate pitching moment model, the locations of the C.Ps. and its movements depending on the pitching velocity were investigated in detail. We found that the aerodynamic moment model became suitable when the rotational force component was assumed to act on the half-chord. This implies that the approximation borrowed from the conventional airfoil concept, i.e., the 'C.P. at the quarter-chord' may lead to an incorrect moment prediction. In the validation process, the model showed excellent time-course force and moment estimations in comparison with the robotic wing measurement results. A fully nonlinear multibody flight dynamic simulation was conducted to check the effect of the traveling C.P. on the overall flight dynamics. This clearly showed the importance of an accurate aerodynamic moment model.

Visfatin, a novel adipokine, stimulates glucose uptake through the Ca2 +-dependent AMPK-p38 MAPK pathway in C2C12 skeletal muscle cells.

Visfatin is a novel adipocytokine produced by visceral fat. In the present study, visfatin increased AMP-activated protein kinase (AMPK) phosphorylation in mouse C2C12 skeletal muscle cells. It also increased phosphorylation of the insulin receptor, whose knockdown blocked visfatin-induced AMPK phosphorylation and glucose uptake. Visfatin stimulated glucose uptake in differentiated skeletal muscle cells. However, inhibition of AMPKα2 with an inhibitor or with knockdown of AMPKα2 using siRNA blocked visfatin-induced glucose uptake, which indicates that visfatin stimulates glucose uptake through the AMPKα2 pathway. Visfatin increased the intracellular Ca(2) (+) concentration. STO-609, a calmodulin-dependent protein kinase kinase inhibitor, blocked visfatin-induced AMPK phosphorylation and glucose uptake. Visfatin-mediated activation of p38 MAPK was AMPKα2-dependent. Furthermore, both inhibition and knockdown of p38 MAPK blocked visfatin-induced glucose uptake. Visfatin increased glucose transporter type 4 (GLUT4) mRNA and protein levels. In addition, visfatin stimulated the translocation of GLUT4 to the plasma membrane, and this effect was suppressed by AMPKα2 inhibition. The present results indicate that visfatin plays an important role in glucose metabolism via the Ca(2) (+)-mediated AMPK-p38 MAPK pathway.

PRMT4 is involved in insulin secretion via the methylation of histone H3 in pancreatic ß cells.

The relationship between protein arginine methyltransferases (PRMTs) and insulin synthesis in ß cells is not yet well understood. In the present study, we showed that PRMT4 expression was increased in INS-1 and HIT-T15 pancreatic ß cells under high-glucose conditions. In addition, asymmetric dimethylation of Arg17 in histone H3 was significantly increased in both cell lines in the presence of glucose. The inhibition or knockdown of PRMT4 suppressed glucose-induced insulin gene expression in INS-1 cells by 81.6 and 79% respectively. Additionally, the overexpression of mutant PRMT4 also significantly repressed insulin gene expression. Consistently, insulin secretion induced in response to high levels of glucose was decreased by both PRMT4 inhibition and knockdown. Moreover, the inhibition of PRMT4 blocked high-glucose-induced insulin gene expression and insulin secretion in primary pancreatic islets. These results indicate that PRMT4 might be a key regulator of high-glucose-induced insulin secretion from pancreatic ß cells via H3R17 methylation.

Irisin, a Novel Myokine, Regulates Glucose Uptake in Skeletal Muscle Cells via AMPK.

Irisin is a novel myokine produced by skeletal muscle. However, its metabolic role is poorly understood. In the present study, irisin induced glucose uptake in differentiated skeletal muscle cells. It increased AMP-activated protein kinase (AMPK) phosphorylation and the inhibition of AMPK blocked glucose uptake. It also increased reactive oxygen species (ROS) generation. N-acetyl cysteine, a ROS scavenger, blocked irisin-induced AMPK phosphorylation. Moreover, irisin activated p38 MAPK in an AMPK-dependent manner. The inhibition and knockdown of p38 MAPK blocked irisin-induced glucose uptake. A colorimetric absorbance assay showed that irisin stimulated the translocation of glucose transporter type 4 to the plasma membrane and that this effect was suppressed in cells pretreated with a p38 MAPK inhibitor or p38 MAPK small interfering RNA. In primary cultured myoblast cells, irisin increased the concentration of intracellular calcium. STO-609, a calcium/calmodulin-dependent protein kinase kinase inhibitor, blocked irisin-induced AMPK phosphorylation, implying that calcium is involved in irisin-mediated signaling. Our results suggest that irisin plays an important role in glucose metabolism via the ROS-mediated AMPK pathway in skeletal muscle cells.

Dibenzoylmethane exerts metabolic activity through regulation of AMP-activated protein kinase (AMPK)-mediated glucose uptake and adipogenesis pathways.

Dibenzoylmethane (DBM) has been shown to exert a variety of beneficial effects on human health. However, the mechanism of action is poorly understood. In this study, DBM increased phosphorylation of AMP-activated protein kinase (AMPK) and stimulated glucose uptake in a skeletal muscle cell line. Both knockdown of AMPK with siRNA and inhibition with AMPK inhibitor blocked DBM-induced glucose uptake. DBM increased the concentration of intracellular calcium and glucose uptake due to DBM was abolished by STO-609 (a calcium/calmodulin-dependent protein kinase inhibitor). DBM stimulated phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), which was blocked by pretreatment with compound C, an AMPK inhibitor. The expression of glucose transporter type 4 (GLUT4) was increased by DBM. The translocation of GLUT4 to the plasma membrane was also increased by DBM in AMPK dependently. In addition, DBM suppressed weight gain and prevented fat accumulation in the liver and abdomen in mice fed a high-fat diet. In pre-adipocyte cells, DBM decreased the activity of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of fatty acid synthesis. Expression of the adipogenic gene, fatty acid synthase (FAS), was suppressed by DBM in an AMPK-dependent manner. These results showed that the beneficial metabolic effects of DBM might be due to regulation of glucose uptake via AMPK in skeletal muscle and inhibition of adipogenesis in pre-adipocytes.

[6]-Gingerol Affects Glucose Metabolism by Dual Regulation via the AMPKα2-Mediated AS160-Rab5 Pathway and AMPK-Mediated Insulin Sensitizing Effects.

[6]-Gingerol has been used to control diabetes and dyslipidemia; however, its metabolic role is poorly understood. In this study, [6]-gingerol increased adenosine monophosphate (AMP)-activated protein kinase (AMPK) phosphorylation in mouse skeletal muscle C2C12 cells. Stimulation of glucose uptake by [6]-gingerol was dependent on AMPKα2. Moreover, both Inhibition and knockdown of AMPKα2 blocked [6]-gingerol-induced glucose uptake. [6]-Gingerol significantly decreased the activity of protein phosphatase 2A (PP2A). Inhibition of PP2A activity with okadaic acid enhanced the phosphorylation of AMPKα2. Moreover, the interaction between AMPKα2 and PP2A was increased by [6]-gingerol, suggesting that PP2A mediates the effect of [6]-gingerol on AMPK phosphorylation. In addition, [6]-gingerol increased the phosphorylation of Akt-substrate 160 (AS160), which is a Rab GTPase-activating protein. Inhibition of AMPKα2 blocked [6]-gingerol-induced AS160 phosphorylation. [6]-gingerol increased the Rab5, and AMPKα2 knockdown blocked [6]-gingerol-induced expression of Rab5, indicating AMPK play as an upstream of Rab5. It also increased glucose transporter 4 (GLUT4) mRNA and protein expression and stimulated GLUT4 translocation. Furthermore, insulin-mediated glucose uptake and Akt phosphorylation were further potentiated by [6]-gingerol treatment. This potentiation was not observed in the presence of AMPK inhibitor compound C. In summary, our results suggest that [6]-gingerol plays an important role in glucose metabolism via the AMPKα2-mediated AS160-Rab5 pathway and through potentiation of insulin-mediated glucose regulation.

A multibody approach for 6-DOF flight dynamics and stability analysis of the hawkmoth Manduca sexta.

This paper investigates the six degrees of freedom (6-DOF) flight dynamics and stability of the hawkmoth Manduca sexta using a multibody dynamics approach that encompasses the effects of the time varying inertia tensor of all the body segments including two wings. The quasi-steady translational and unsteady rotational aerodynamics of the flapping wings are modeled with the blade element theory with aerodynamic coefficients derived from relevant experimental studies. The aerodynamics is given instantaneously at each integration time step without wingbeat-cycle-averaging. With the multibody dynamic model and the aerodynamic model for the hawkmoth, a direct time integration of the fully coupled 6-DOF nonlinear multibody dynamics equations of motion is performed. First, the passive damping magnitude of each single DOF is quantitatively examined with the measure of the time taken to half the initial velocity (thalf). The results show that the sideslip translation is less damped approximately three times than the other two translational DOFs, and the pitch rotation is less damped approximately five times than the other two rotational DOFs; each DOF has the value of (unit in wingbeat strokes): thalf,forward/backward = 7.10, thalf,sideslip = 17.95, thalf,ascending = 7.13, thalf,descending = 5.77, thalf,roll = 0.68, thalf,pitch = 2.39, and thalf,yaw = 0.25. Second, the natural modes of motion, with the hovering flight as a reference equilibrium condition, are examined by analyzing fully coupled 6-DOF dynamic responses induced by multiple sets of force and moment disturbance combinations. The given disturbance combinations are set to excite the dynamic modes identified in relevant eigenmode analysis studies. The 6-DOF dynamic responses obtained from this study are compared with eigenmode analysis results in the relevant studies. The longitudinal modes of motion showed dynamic modal characteristics similar to the eigenmode analysis results from the relevant literature. However, the lateral modes of motion revealed more complex behavior, which is mainly due to the coupling effect in the lateral flight states and also between the lateral and longitudinal planes of motion. The main sources of the flight instability of the hovering hawkmoth are examined as either the longitudinal instability grown from the coupled forward/backward velocity and the pitch rate, or the lateral instability grown from the coupled sideslip velocity and the roll rate.