Hiroyasu Hatakeyama

Palmitate-induced down-regulation of sortilin and impaired GLUT4 trafficking in C2C12 myotubes

Elevated saturated FFAs including palmitate (C16:0) are a primary trigger for peripheral insulin resistance characterized by impaired glucose uptake/disposal in skeletal muscle, resulting from impaired GLUT4 translocation in response to insulin. We herein demonstrate that palmitate induces down-regulation of sortilin, a sorting receptor implicated in the formation of insulin-responsive GLUT4 vesicles, via mechanisms involving PKCθ and TNF-α-converting enzyme, but not p38, JNK, or mitochondrial reactive oxygen species generation, leading to impaired GLUT4 trafficking in C2C12 myotubes. Intriguingly, unsaturated FFAs such as palmitoleate (C16:1) and oleate (C18:1) had no such detrimental effects, appearing instead to effectively reverse palmitate-induced impairment of insulin-responsive GLUT4 recycling along with restoration of sortilin abundance by preventing aberrant PKCθ activation. On the other hand, shRNA-mediated reduction of sortilin in intact C2C12 myotubes inhibited insulin-induced GLUT4 recycling without dampening Akt phosphorylation. We found that the peroxisome proliferator-activated receptor γ agonist troglitazone prevented the palmitate-induced sortilin reduction and also ameliorated insulin-responsive GLUT4 recycling without altering the palmitate-evoked insults on signaling cascades; neither highly phosphorylated PKCθ states nor impaired insulin-responsive Akt phosphorylation was affected. Taken together, our data provide novel insights into the pathogenesis of PKCθ-dependent insulin resistance with respect to insulin-responsive GLUT4 translocation, which could occur not only through defects of insulin signaling but also via a reduction of sortilin, which directly controls trafficking/sorting of GLUT4 in skeletal muscle cells. In addition, our data suggest the insulin-sensitizing action of peroxisome proliferator-activated receptor γ agonists to be at least partially mediated through the restoration of proper GLUT4 trafficking/sorting events governed by sortilin.

Identification of Three Distinct Functional Sites of Insulin-mediated GLUT4 Trafficking in Adipocytes Using Quantitative Single Molecule Imaging

We developed a novel approach allowing intracellular GLUT4 dynamics to be analyzed directly at the single molecule level using Quantum dot to quantitatively establish the behavioral nature of GLUT4. With this approach, we defined the actual steps at which insulin signals directly converge and impact the process of dynamic GLUT4 trafficking events.

Characterization of contraction-inducible CXC chemokines and their roles in C2C12 myocytes

Physical exercise triggers the release of several cytokines/chemokines from working skeletal muscles, but the underlying mechanism(s) by which skeletal muscles decipher and respond to highly complex contractile stimuli remains largely unknown. In an effort to investigate the regulatory mechanisms of the expressions of two contraction-inducible CXC chemokines, CXCL1/KC and CXCL5/LIX, in contracting skeletal muscle cells, we took advantage of our in vitro exercise model using highly developed contractile C(2)C(12) myotubes, which acquire properties similar to those of in vivo skeletal muscle via manipulation of Ca(2+) transients with electric pulse stimulation (EPS). Production of these CXC chemokines was immediately augmented by EPS-evoked contractile activity in a manner dependent on the activities of JNK and NF-kappaB, but not p38, ERK1/2, or calcineurin. Intriguingly, exposure of myotubes to cyclic mechanical stretch also induced expression of these CXC chemokines; however, a much longer period of stimulation (approximately 12 h) was required, despite rapid JNK phosphorylation. We also demonstrate herein that CXCL1/KC and CXCL5/LIX have the ability to raise intracellular Ca(2+) concentrations via CXCR2-mediated activation of pertussis toxin-sensitive Galpha(i) proteins in C(2)C(12) myoblasts, an action at least partially responsible for their migration and differentiation. Although we revealed a possible negative feedback regulation of their own production in response to the contractile activity in differentiated myotubes, exogenous administration of these CXC chemokines did not acutely influence either insulin-induced Akt phosphorylation or GLUT4 translocation in C(2)C(12) myotubes. Taken together, these data shed light on the fundamental characteristics of contraction-inducible CXC chemokine production and their potential roles in skeletal muscle cells.

Different impacts of saturated and unsaturated free fatty acids on COX-2 expression in C2C12 myotubes

In skeletal muscle, saturated free fatty acids (FFAs) act as proinflammatory stimuli, and cyclooxygenase-2 (COX-2) is a pro/anti-inflammatory enzyme induced at sites of inflammation, which contributes to prostaglandin production. However, little is known about the regulation of COX-2 expression and its responses to FFAs in skeletal muscle. Herein, we examined the effects of saturated and unsaturated FFAs, including a recently identified lipokine (lipid hormone derived from adipocytes), palmitoleate, on COX-2 expression in C(2)C(12) myotubes as a skeletal muscle model. Exposure of myotubes to saturated FFAs [palmitate (16:0) and stearate (18:0)], but not to unsaturated FFAs [palmitoleate (16:1), oleate (18:1), and linoleate (18:2)], led to a slow-onset induction of COX-2 expression and subsequent prostaglandin E(2) production via mechanisms involving the p38 MAPK and NF-kappaB but not the PKC signaling cascades. Pharmacological modulation of mitochondrial oxidative function failed to interfere with COX-2 expression, suggesting the mitochondrial overload/excessive beta-oxidation contribution to this event to be minimal. On the contrary, unsaturated FFAs appeared to effectively antagonize palmitate-induced COX-2 expression with markedly different potencies (linoleate > oleate > palmitoleate), being highly associated with the suppressive profile of each unsaturated FFA toward palmitate-evoked intracellular signals, including p38, JNK, ERK1/2 MAPKs, and PKCtheta, as well as IkappaB degradation. In addition, our data suggest little involvement of PPAR in the protective actions of unsaturated FFAs against palmitate-induced COX-2 expression. No direct contribution of the increased COX-2 activity in generating palmitate-induced insulin resistance was detected, at least in terms of insulin-responsive Akt phosphorylation and GLUT4 translocation. Taken together, our data provide a novel insight into the molecular mechanisms responsible for the FFA-induced COX-2 expression in skeletal muscle and raise the possibility that, in skeletal myocytes, COX-2 and its product prostaglandins may play an important role in the complex inflammation responses caused by elevated FFAs, for example, in the diabetic state.

Molecular Basis of Insulin‐Responsive GLUT4 Trafficking Systems Revealed by Single Molecule Imaging

Development of a ‘static retention’ property of GLUT4, the insulin‐responsive glucose transporter, has emerged as being essential for achieving its maximal insulin‐induced surface exposure. Herein, employing quantum‐dot‐based nanometrology of intracellular GLUT4 behavior, we reveal the molecular basis of its systematization endowed upon adipogenic differentiation of 3T3L1 cells. Specifically, (i) the endosomes‐to‐trans‐Golgi network (TGN) retrieval system specialized for GLUT4 develops in response to sortilin expression, which requires an intricately balanced interplay among retromers, golgin‐97 and syntaxin‐6, the housekeeping vesicle trafficking machinery. (ii) The Golgin‐97‐localizing subdomain of the differentiated TGN apparently serves as an intermediate transit route by which GLUT4 can further proceed to the stationary GLUT4 storage compartment. (iii) AS160/Tbc1d4 then renders the ‘static retention’ property insulin responsive, i.e. insulin liberates GLUT4 from the static state only in the presence of functional AS160/Tbc1d4. (iv) Moreover, sortilin malfunction and the resulting GLUT4 sorting defects along with retarded TGN function might be etiologically related to insulin resistance. Together, these observations provide a conceptual framework for understanding maturation/retardation of the insulin‐responsive GLUT4 trafficking system that relies on the specialized subdomain of differentiated TGN.

Regulatory mode shift of Tbc1d1 is required for acquisition of insulin-responsive GLUT4-trafficking activity.

Tbc1d1 is involved in AICAR-dependent GLUT4 liberation. Tbc1d1 acquires temporal insulin responsiveness with AICAR pretreatment. This shift in regulatory mode requires Ser- 237 phosphorylation and the PTB1 domain. PTB1 mutants exhibit no shift in regulatory mode and thus no insulin responsiveness.

Single quantum dot tracking reveals that an individual multivalent HIV-1 Tat protein transduction domain can activate machinery for lateral transport and endocytosis.

ABSTRACT The mechanisms underlying the cellular entry of the HIV-1 Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatPs behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cells lateral transport machinery to move the mTatP-QD toward the center of the cell body upon cross-linking of heparan sulfate proteoglycans. The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic internalization. However, bivalent TatP did not sufficiently promote either cell surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo delivery nanomachines.

Live-cell single-molecule labeling and analysis of myosin motors with quantum dots

Quantum dots (QDs) are a powerful tool for quantitative biology, but two challenges are associated with using them to track intracellular molecules in live cells. A simple and convenient method is presented for labeling intracellular molecules by using HaloTag technology and electroporation and is used to successfully track myosins within live cells.

Development of dual-color simultaneous single molecule imaging system for analyzing multiple intracellular trafficking activities

Intracellular trafficking is a critical process for cell physiology. Previous extensive studies employing biochemical and molecular biological approaches have provided qualitative information about intracellular trafficking, but we have little quantitative information due to technical limitations of these assays. We therefore developed a novel method for quantifying intracellular trafficking based on single molecule imaging with Quantum dot (QD) fluorescent nanocrystals and quantitatively described the trafficking properties of some recycling proteins. We herein first describe how to label intracellular molecules with QD which has no cell permeability and how to quantify intracellular trafficking, and then we detail the development of a novel experimental system allowing multi-color simultaneous single molecule imaging for analyzing the relationships of intracellular trafficking activities among multiple molecules having distinct trafficking properties. Finally, we document how we confirmed the reliability of our system by simultaneously analyzing the intracellular movements of two recycling protein, GLUT4 glucose transporter and transferrin receptor. Since impairment of intracellular trafficking has critical etiological roles in various late-onset diseases such as type 2 diabetes, our novel imaging system may be a powerful tool for developing next-generation biomedical devices for diagnostics and medical treatment based on intracellular trafficking.

Heterotypic endosomal fusion as an initial trigger for insulin‐induced glucose transporter 4 (GLUT4) translocation in skeletal muscle

Comprehensive imaging analyses of glucose transporter 4 (GLUT4) behaviour in mouse skeletal muscle was conducted. Quantum dot‐based single molecule nanometry revealed that GLUT4 molecules in skeletal myofibres are governed by regulatory systems involving ‘static retention’ and ‘stimulus‐dependent liberation’. Vital imaging analyses and super‐resolution microscopy‐based morphometry demonstrated that insulin liberates the GLUT4 molecule from its static state by triggering acute heterotypic endomembrane fusion arising from the very small GLUT4‐containing vesicles in skeletal myofibres. Prior exposure to exercise‐mimetic stimuli potentiated this insulin‐responsive endomembrane fusion event involving GLUT4‐containing vesicles, suggesting that this endomembranous regulation process is a potential site related to the effects of exercise.