Padmamalini Baskaran

Capsaicin induces browning of white adipose tissue and counters obesity by activating TRPV1 channel‐dependent mechanisms

The growing epidemic of obesity and metabolic diseases necessitates the development of novel strategies to prevent and treat such diseases. Current research suggests that browning of white adipose tissue (WAT) promotes energy expenditure to counter obesity. Recent research suggests that activation of the TRPV1 channels counters obesity. However, the mechanism by which activation of TRPV1 channels counters obesity still remains unclear.

The TRPM8 Protein Is a Testosterone Receptor II. FUNCTIONAL EVIDENCE FOR AN IONOTROPIC EFFECT OF TESTOSTERONE ON TRPM8

Background: TRPM8 channels are highly expressed in prostate tissues, where the role of this cold receptor is not well understood. Results: Testosterone activates TRPM8 in various cellular systems and in the planar lipid bilayers. Conclusion: TRPM8 is an ionotropic testosterone receptor. Significance: TRPM8 channels may be implicated in various physiological processes regulated by androgens. Testosterone is a key steroid hormone in the development of male reproductive tissues and the regulation of the central nervous system. The rapid signaling mechanism induced by testosterone affects numerous behavioral traits, including sexual drive, aggressiveness, and fear conditioning. However, the currently identified testosterone receptor(s) is not believed to underlie the fast signaling, suggesting an orphan pathway. Here we report that an ion channel from the transient receptor potential family, TRPM8, commonly known as the cold and menthol receptor is the major component of testosterone-induced rapid actions. Using cultured and primary cell lines along with the purified TRPM8 protein, we demonstrate that testosterone directly activates TRPM8 channel at low picomolar range. Specifically, testosterone induced TRPM8 responses in primary human prostate cells, PC3 prostate cancer cells, dorsal root ganglion neurons, and hippocampal neurons. Picomolar concentrations of testosterone resulted in full openings of the purified TRPM8 channel in planar lipid bilayers. Furthermore, acute applications of testosterone on human skin elicited a cooling sensation. Our data conclusively demonstrate that testosterone is an endogenous and highly potent agonist of TRPM8, suggesting a role of TRPM8 channels well beyond their well established function in somatosensory neurons. This discovery may further imply TRPM8 channel function in testosterone-dependent behavioral traits.

The TRPM8 Protein Is a Testosterone Receptor I. BIOCHEMICAL EVIDENCE FOR DIRECT TRPM8-TESTOSTERONE INTERACTIONS

Background: TRPM8 channels are highly expressed in prostate tissues, where the role of this cold receptor is not well understood. Results: Testosterone directly interacts with the TRPM8 protein. Conclusion: TRPM8 is a testosterone receptor. Significance: TRPM8 channels may be implicated in various physiological processes regulated by androgens. The transient receptor potential ion channel of the melastatin subfamily, TRPM8, is a major cold receptor in the peripheral nervous system. Along with the sensory neurons, the TRPM8 protein is highly expressed in the prostate epithelial cells, and this expression is regulated by androgens. Here we investigated the expression and intracellular localization of the TRPM8 channel in relationship to androgens. We performed experiments using human prostate tissues obtained from healthy individuals and patients with prostate cancer at various stages of the disease as well as in cultured cells. Using an immunohistochemistry approach, we detected an intensive colocalization pattern of the TRPM8 protein with endogenous androgens in all tissues tested, suggesting possible interactions. Co-immunoprecipitation experiments performed using cultured prostate epithelial cells, prostate cancer cells, and HEK-293 cells stably expressing TRPM8 further confirmed direct binding of the steroid hormone, testosterone, to the TRPM8 protein. Applications of picomolar concentrations of testosterone to the primary human prostate cells, endogenously expressing TRPM8, elicited Ca2+ responses and channel currents, and those were inhibited in the presence of TRPM8 antagonist, N-(2-aminoethyl)-N-(4-(benzyloxy)-3-methoxybenzyl)thiophene-2-carboxamide hydrochloride. These results indicate that the TRPM8 channel is physically associated with testosterone and suggest that, in addition to a genomic role, testosterone plays a role in direct regulation of the TRPM8 channel function.

Cyclophilin A Is Required for Angiotensin II–Induced p47phox Translocation to Caveolae in Vascular Smooth Muscle Cells

Objective—Angiotensin II (AngII) signal transduction in vascular smooth muscle cells (VSMC) is mediated by reactive oxygen species (ROS). Cyclophilin A (CyPA) is a ubiquitously expressed cytosolic protein that possesses peptidyl-prolyl cis-trans isomerase activity, scaffold function, and significantly enhances AngII-induced ROS production in VSMC. We hypothesized that CyPA regulates AngII-induced ROS generation by promoting translocation of NADPH oxidase cytosolic subunit p47phox to caveolae of the plasma membrane. Approach and Results—Overexpression of CyPA in CyPA-deficient VSMC (CyPA−/−VSMC) significantly increased AngII-stimulated ROS production. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors (VAS2870 or diphenylene iodonium) significantly attenuated AngII-induced ROS production in CyPA and p47phox-overexpressing CyPA−/−VSMC. Cell fractionation and sucrose gradient analyses showed that AngII-induced p47phox plasma membrane translocation, specifically to the caveolae, was reduced in CyPA−/−VSMC compared with wild-type–VSMC. Immunofluorescence studies demonstrated that AngII increased p47phox and CyPA colocalization and translocation to the plasma membrane. In addition, immunoprecipitation of CyPA followed by immunoblotting of p47phox and actin showed that AngII increased CyPA and p47phox interaction. AngII-induced p47phox and actin cell cytoskeleton association was attenuated in CyPA−/−VSMC. Mechanistically, inhibition of p47phox phosphorylation and phox homology domain deletion attenuated CyPA and p47phox interaction. Finally, cyclosporine A and CyPA-peptidyl-prolyl cis-trans isomerase mutant, R55A, inhibited AngII-stimulated CyPA and p47phox association in VSMC, suggesting that peptidyl-prolyl cis-trans isomerase activity was required for their interaction. Conclusions—These findings provide the mechanism by which CyPA is an important regulator for AngII-induced ROS generation in VSMC through interaction with p47phox and cell cytoskeleton, which enhances the translocation of p47phox to caveolae.

Acetylation of cyclophilin A is required for its secretion and vascular cell activation

AIMS Cyclophilin A (CyPA) is a pro-inflammatory mediator involved in oxidative stress-related cardiovascular diseases. It is secreted from vascular smooth muscle cell (VSMC) in response to reactive oxygen species (ROS) in a highly regulated manner. Extracellular CyPA activates VSMCs and endothelial cells (ECs) promoting inflammation, cell growth, and cell death. Recently, it was shown that acetylated CyPA (AcK-CyPA) affects its function. We investigated the role of acetylation of CyPA for its secretion and signalling in vascular cells. METHODS AND RESULTS We used angiotensin II (Ang II) to create sustained ROS and found significantly increased AcK-CyPA in VSMC. Site-directed mutagenesis showed that lysines K82 and K125 were the predominant CyPA residues acetylated in response to Ang II. Importantly, acetylation of K82 and K125 were required for Ang II-mediated CyPA secretion. ROS inhibitors, Tiron, and N-acetylcysteine inhibited Ang II-induced intracellular CyPA acetylation and also AcK-CyPA secretion. Using secreted CyPA from wild type and K82/125R mutants expressed in transduced VSMC or in vitro acetylated recombinant CyPA, we showed that extracellular AcK-CyPA significantly increased pERK1/2, matrix metalloproteinase-2 activation, and ROS production in VSMC compared with non-acetylated CyPA. Moreover, extracellular AcK-CyPA increased adhesion molecule expression (VCAM-1 and ICAM-1) in EC, which promoted monocyte adhesion. CONCLUSIONS ROS-dependent acetylation of CyPA is required for the generation of extracellular CyPA. Acetylated extracellular CyPA regulates VSMC and EC activation, suggesting that inhibition of acetylation of CyPA may prevent the pathogenesis of oxidative stress-related cardiovascular diseases.

TRPV1 activation counters diet-induced obesity through sirtuin-1 activation and PRDM-16 deacetylation in brown adipose tissue

Background/objective:An imbalance between energy intake and expenditure leads to obesity. Increasing metabolism and thermogenesis in brown adipose tissue (BAT) can help in overcoming obesity. Here, we investigated the effect of activation of transient receptor potential vanilloid subfamily 1 (TRPV1) in the upregulation of thermogenic proteins in BAT to counter diet-induced obesity.Subjects/methods:We investigated the effect of dietary supplementation of capsaicin (CAP) (TRPV1 agonist) on the expression of metabolically important thermogenic proteins in BAT of wild-type and TRPV1−/− mice that received either a normal chow or high-fat (±CAP; TRPV1 activator) diet by immunoblotting. We measured the metabolic activity, respiratory quotient and BAT lipolysis.Results:CAP antagonized high-fat diet (HFD)-induced obesity without decreasing energy intake in mice. HFD suppressed TRPV1 expression and activity in BAT and CAP countered this effect. HFD-feeding caused glucose intolerance, hypercholesterolemia and decreased the plasma concentration of glucagon-like peptide-1 and CAP countered these effects. HFD suppressed the expression of metabolically important thermogenic genes, ucp-1, bmp8b, sirtuin-1 (SIRT-1), PPARγ coactivator 1α and PR domain containing zinc finger protein 16 (prdm-16) in BAT and CAP prevented this effect. CAP increased the phosphorylation of SIRT-1 and induced an interaction between peroxisome proliferator activated receptor gamma (PPARγ) with PRDM-16. Further, CAP treatment, in vitro, decreased the acetylation of PRDM-16, which was antagonized by inhibition of TRPV1 by capsazepine, chelation of intracellular Ca2+ by cell permeable BAPTA-AM or the inhibition of SIRT-1 by EX527. Further, CAP supplementation, post HFD, promoted weight loss and enhanced the respiratory exchange ratio. CAP did not have any effect in TRPV1−/− mice.Conclusions:Our data show that activation of TRPV1 in BAT enhances the expression of SIRT-1, which facilitates the deacetylation and interaction of PPARγ and PRDM-16. These data suggest that TRPV1 activation is a novel strategy to counter diet-induced obesity by enhancing metabolism and energy expenditure.

Capsaicin modulates acetylcholine release at the myoneural junction.

Transient receptor potential (TRP) proteins are non-selective cation channel proteins that are expressed throughout the body. Previous studies demonstrated the expression of TRP Vanilloid 1 (TRPV1), capsaicin (CAP) receptor, in sensory neurons. Recently, we reported TRPV1 expression in mouse motor nerve terminals [MNTs; (Thyagarajan et al., 2009)], where we observed that CAP protected MNTs from botulinum neurotoxin A (BoNT/A). Phrenic nerve diaphragm nerve muscle preparations (NMP) isolated from isoflurane anesthetized adult mice were analyzed for twitch tension, spontaneous (mEPCs) and nerve stimulus evoked (EPCs) acetylcholine release. When acutely applied to isolated NMP, CAP produced a concentration-dependent decline of twitch tension and produced a significant decline in the amplitude of EPCs and quantal content without any effect on the mEPCs. The suppression of nerve stimulus evoked acetylcholine release by CAP was antagonized by capsazepine (CPZ), a TRPV1 antagonist. CAP did not suppress phrenic nerve stimulus evoked acetylcholine release in TRPV1 knockout mice. Also, CAP treatment, in vitro, interfered with the localization of adapter protein 2 in cholinergic Neuro 2a cells. Wortmannin, (WMN; non-selective phosphoinositol kinase inhibitor), mimicked the effects of CAP by inhibiting the acetylcholine exocytosis. Our data suggest that TRPV1 proteins expressed at the MNT are coupled to the exo-endocytic mechanisms to regulate neuromuscular functions.

Acute and chronic effects of botulinum neurotoxin a on the mammalian neuromuscular junction

Botulinum neurotoxin A (BoNT/A) cleaves SNAP‐25 and inhibits acetylcholine (ACh) release at the neuromuscular junctions (NMJ) to cause neuroparalysis. Previous reports indicate a dyssynchrony between the inhibitory effect of BoNT/A on ACh release and SNAP‐25 cleavage. Methods: We tested the in vitro (acute; 90 min) and in vivo (chronic; 12 h) effects of BoNT/A on stimulus‐evoked ACh release (SEAR), twitch tension, and SNAP‐25 cleavage in isolated extensor digitorum longus (EDL) nerve‐muscle preparations (NMP).Results: In vitro or in vivo BoNT/A poisoning inhibited SEAR and twitch tension. Conversely, SNAP‐25 cleavage and inhibition of spontaneous release frequency were observed only in NMP poisoned with BoNT/A in vivo. Moreover, chronic treatment of BoNT/A inhibited ionomycin stimulated Ca2+ signals in Neuro 2a cells. Conclusions: These results demonstrate that the inhibition of SEAR precedes SNAP‐25 cleavage and suggest involvement of a more complex mechanism for the inhibitory effect of BoNT/A at the NMJ. Muscle Nerve 50:206–215, 2014

Perturbation to Cholesterol at the Neuromuscular Junction Confers Botulinum Neurotoxin A Sensitivity to Neonatal Mice

Botulinum neurotoxin A (BoNT/A) cleaves SNAP25 at the motor nerve terminals and inhibits stimulus evoked acetylcholine release. This causes skeletal muscle paralysis. However, younger neonatal mice (<P7; <7-days old) are resistant to the neuroparalytic effects of BoNT/A. That is, invivo injection of BoNT/A at the innervations of Extensor digitorum longus muscle in the hindlimbs inhibited the toe spread reflex within 24 hours following BoNT/A injection in adult mouse and in older (>P7) mice. However, neonatal mice younger than 7 days-age remained unaffected by BoNT/A injection. Also, BoNT/A inhibited stimulus evoked acetylcholine release and stimulus-evoked twitch tension of diaphragm nerve muscle preparations (NMPs) of adult mouse and >P7 neonates but not that of <P7. Moreover, NMPs of <P7 showed decreased uptake of fluorescent BoNT/A compared to >P7. However, cholesterol depletion using methyl-β-cyclodextrin (MβCD) sensitized <P7 neonates to BoNT/A and facilitated BoNT/A uptake into NMPs obtained from <P7 neonates. Further, MβCD (10 mM; 30 min pretreatment) increased the interaction between synaptic vesicle protein 2 and BoNT/A. Also, cholesterol depletion increased the miniature endplate current in adult NMPs. Interestingly, cholesterol replenishment, invitro, delayed the onset of inhibitory effect of BoNT/A. Collectively, our data suggest that cholesterol rich lipid microdomains are involved in BoNT/A uptake mechanisms during development. Our data demonstrate that cholesterol depletion sensitized neonatal mice (<P7) to BoNT/A while replenishing cholesterol delayed the onset of inhibitory actin of BoNT/A. This suggests that membrane cholesterol modulates neurotoxin sensitivity at the neuromuscular junction (NMJ).

Measurement of Basal and Forskolin-stimulated Lipolysis in Inguinal Adipose Fat Pads

Lipolysis is a process by which the lipid stored as triglycerides in adipose tissues are hydrolyzed into glycerol and fatty acids. This article describes the method for the measurement of basal and forskolin (FSK)-stimulated lipolysis in the inguinal fat pads isolated from wild type mice fed either normal chow diet (NCD), high fat diet (HFD) or a high fat diet containing 0.01% of capsaicin (CAP; transient receptor potential vanilloid subfamily 1 (TRPV1) agonist) for 32 weeks. The method described here for performing ex vivo lipolysis is adopted from Schweiger et al.1 We present a detailed protocol for measuring glycerol levels by UV-Visible (UV/VIS) spectrophotometry. The method described here can be used to successfully isolate inguinal fat pads for lipolysis measurements to obtain consistent results. The protocol described for inguinal fat pads can readily be extended to measure lipolysis in other tissues.