Activation of Toll-like Receptor 4 (TLR4) Attenuates Adaptive Thermogenesis via Endoplasmic Reticulum Stress

Adaptive thermogenesis is the cellular process transforming chemical energy into heat in response to cold. A decrease in adaptive thermogenesis is a contributing factor to obesity. However, the molecular mechanisms responsible for the compromised adaptive thermogenesis in obese subjects have not yet been elucidated. In this study we hypothesized that Toll-like receptor 4 (TLR4) activation and subsequent inflammatory responses are key regulators to suppress adaptive thermogenesis. To test this hypothesis, C57BL/6 mice were either fed a palmitate-enriched high fat diet or administered with chronic low-dose LPS before cold acclimation. TLR4 stimulation by a high fat diet or LPS were both associated with reduced core body temperature and heat release. Impairment of thermogenic activation was correlated with diminished expression of brown-specific markers and mitochondrial dysfunction in subcutaneous white adipose tissue (sWAT). Defective sWAT browning was concomitant with elevated levels of endoplasmic reticulum (ER) stress and autophagy. Consistently, TLR4 activation by LPS abolished cAMP-induced up-regulation of uncoupling protein 1 (UCP1) in primary human adipocytes, which was reversed by silencing of C/EBP homologous protein (CHOP). Moreover, the inactivation of ER stress by genetic deletion of CHOP or chemical chaperone conferred a resistance to the LPS-induced suppression of adaptive thermogenesis. Collectively, our data indicate the existence of a novel signaling network that links TLR4 activation, ER stress, and mitochondrial dysfunction, thereby antagonizing thermogenic activation of sWAT. Our results also suggest that TLR4/ER stress axis activation may be a responsible mechanism for obesity-mediated defective brown adipose tissue activation.     

Expression of the Cameleon calcium biosensor in fungi reveals distinct Ca2+ signatures associated with polarized growth,development, and pathogenesis

Calcium is a universal messenger that translates diverse environmental stimuli and developmental cues into specific cellular and developmental responses. While individual fungal species have evolved complex and often unique biochemical and structural mechanisms to exploit specific ecological niches and to adjust growth and development in response to external stimuli, one universal feature to all is that Ca²⁺-mediated signaling is involved. The lack of a robust method for imaging spatial and temporal dynamics of subcellular Ca²⁺ (i.e., “Ca²⁺ signature”), readily available in the plant and animal systems, has severely limited studies on how this signaling pathway controls fungal growth, development, and pathogenesis. Here, we report the first successful expression of a FRET (Förster Resonance Energy Transfer)-based Ca²⁺ biosensor in fungi. Time-lapse imaging of Magnaporthe oryzae, Fusarium oxysporum, and Fusarium graminearum expressing this sensor showed that instead of a continuous gradient, the cytoplasmic Ca²⁺ ([Ca²⁺]_c) change occurred in a pulsatile manner with no discernable gradient between pulses, and each species exhibited a distinct Ca²⁺ signature. Furthermore, occurrence of pulsatile Ca²⁺ signatures was age and development dependent, and major [Ca²⁺]_c transients were observed during hyphal branching, septum formation, differentiation into specialized plant infection structures, cell–cell contact and in planta growth. In combination with the sequenced genomes and ease of targeted gene manipulation of these and many other fungal species, the data, materials and methods developed here will help understand the mechanism underpinning Ca²⁺-mediated control of cellular and developmental changes, its role in polarized growth forms and the evolution of Ca²⁺ signaling across eukaryotic kingdoms.     

Down-regulation of aldose reductase renders J774A.1 cells more susceptible to acrolein- or hydrogen peroxide-induced cell death

Aldose reductase (AR) is abundantly expressed in a variety of cell lineages and has been implicated in the cellular response against oxidative stress. However, the exact functional role of AR against oxidative stress remains relatively unclear. This study investigated the role of AR in acrolein- or hydrogen peroxide-induced apoptosis using the J774.A.1 macrophage cell line. Ablation of AR with a small interference RNA or inhibition of AR activity significantly enhanced the acrolein- or hydrogen peroxide-induced generation of reactive oxygen species and aldehydes, leading to increased apoptotic cell death. Blockade of AR activity in J774A.1 cells markedly augmented the acrolein- or hydrogen peroxide-induced translocation of Bax to mitochondria along with reduced Bcl-2 and increased release of cytochrome c from the mitochodria. Taken together, these findings indicate that AR plays an important role in the cellular response against oxidative stress, by sequestering the reactive molecules generated in cells exposed to toxic substances.     

Single-channel properties and pH sensitivity of two-pore domain K+ channels of the TALK family

The two-pore K2P channel family comprises TASK, TREK, TWIK, TRESK, TALK, and THIK subfamilies, and TALK-1, TALK-2, and TASK-2 are functional members of the TALK subfamily. Here we report for the first time the single-channel properties of TALK-2 and its pHo sensitivity, and compare them to those of TALK-1 and TASK-2. In transfected COS-7 cells, the three TALK K2P channels could be identified easily by their differences in single-channel conductance and gating kinetics. The single-channel conductances of TALK-1, TALK-2, and TASK-2 in symmetrical 150 mM KCl were 21, 33, and 70 pS (-60 mV), respectively. TALK-2 was sensitive mainly to the alkaline range (pH 7-10), whereas TALK-1 and TASK-2 were sensitive to a wider pHo range (6-10). The effect of pH changes was mainly on the opening frequency. Thus, members of the TALK family expressed in native tissues may be identified based on their single-channel kinetics and pHo sensitivity.     

Triptolide inhibits colon cancer cell proliferation and induces cleavage and translocation of 14‐3‐3 epsilon

Triptolide is a diterpenoid triepoxide derived from the traditional Chinese medical herb Tripterygium wilfordii. In the present study, we demonstrated that this phytochemical attenuated colon cancer growth in vitro and in vivo. Using a proteomic approach, we found that 14‐3‐3 epsilon, a cell cycle‐ and apoptosis‐related protein, was altered in colon cancer cells treated with triptolide. In this regard, triptolide induced cleavage and perinuclear translocation of 14‐3‐3 epsilon. Taken together, our findings suggest that triptolide may merit investigation as a potential therapeutic agent for colon cancer, and its anticancer action may be associated with alteration of 14‐3‐3 epsilon. Copyright © 2012 John Wiley & Sons, Ltd.