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Epidermal energy metabolism is relevant to skin physiology, ageing and photodamage. While selected hormones stimulate epidermal keratinocyte mitochondrial activity, its negative regulation remains unknown. In several cell types, cannabinoid receptor 1 (CB1 ) is expressed both on the cell membrane (cmCB1 ) and on the mitochondrial outer membrane (mtCB1 ), where its stimulation directly suppresses mitochondrial functions. In the current pilot study, we investigated if CB1 is a negative regulator of human epidermal energy metabolism under physiological conditions. Using organ-cultured full-thickness human skin specimens of healthy individuals, we showed that antagonizing the homeostatic CB1 signalling by the administration of the CB1 inverse agonist AM251 increased respiratory chain complex I and II/IV activity. The effect was CB1 -dependent, since the CB1 -selective agonist arachidonyl-2'-chloroethylamide could prevent the effect. Moreover, the phenomenon was also reproduced by siRNA-mediated down-regulation of CB1 . As revealed by the unaltered expression of several relevant markers (TFAM, VDAC1, MTCO1 and NDUFS4), modulation of CB1 signalling had no effect on the epidermal mitochondrial mass. Next, by using immunoelectron microscopy, we found that human epidermal keratinocytes express both cmCB1 and mtCB1 . Finally, by using equipotent extracellularly restricted (hemopressin) as well as cell-permeable (AM251) inverse agonists, we found that mitochondrial activity is most likely exclusively regulated by mtCB1 . Thus, our data identify mtCB1 as a novel negative regulator of keratinocyte mitochondrial activity in intact human epidermis, and raise the question, whether topical therapeutic interventions capable of selectively activating mtCB1 can reduce excessive mitochondrial ROS production resulting from dysregulated mitochondrial activity during skin ageing or photodamage.

Background and Purpose Glycogen phosphorylase (GP) is the key enzyme for glycogen degradation. GP inhibitors (GPi-s) are glucose lowering agents that cause the accumulation of glucose in the liver as glycogen. Glycogen metabolism has implications in beta cell function. Glycogen degradation can maintain cellular glucose levels, which feeds into catabolism to maintain insulin secretion, and elevated glycogen degradation levels contribute to glucotoxicity. The purpose of this study was to assess whether influencing glycogen metabolism in beta cells by GPi-s affects the function of these cells. Experimental Approach The effects of structurally different GPi-s were investigated on MIN6 insulinoma cells and in a mouse model of diabetes. Key Results GPi treatment increased glycogen content and, consequently, the surface area of glycogen in MIN6 cells. Furthermore, GPi treatment induced insulin receptor β (InsRβ), Akt and p70S6K phosphorylation, as well as pancreatic and duodenal homeobox 1(PDX1) and insulin expression. In line with these findings, GPi-s enhanced non-stimulated and glucose-stimulated insulin secretion in MIN6 cells. The InsRβ was shown to co-localize with glycogen particles as confirmed by in silico screening, where components of InsR signalling were identified as glycogen-bound proteins. GPi-s also activated the pathway of insulin secretion, indicated by enhanced glycolysis, mitochondrial oxidation and calcium signalling. Finally, GPi-s increased the size of islets of Langerhans and improved glucose-induced insulin release in mice. Conclusion and Implications These data suggest that GPi-s also target beta cells and can be repurposed as agents to preserve beta cell function or even ameliorate beta cell dysfunction in different forms of diabetes. Linked Articles This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc

One of the most abundant coagulation proteins is β2-glycoprotein I (β2GPI) that is present in humans at a concentration of around 200 mg/L. Its physiological role is only partially understood, but it adopts several different structural forms the majority of which are the open and closed forms. We isolated native (circular) β2GPI and converted it into an open conformation. The effectiveness of these procedures was assessed by Western blot and negative-staining electron microscopy. We found that in coagulation assays the open form of β2GPI had a significant prolonging effect on fibrin formation in a dilute prothrombin time test (p

Poly(ADP-Ribose) polymerases (PARPs) are enzymes that metabolize NAD+. PARP1 and PARP10 were previously implicated in the regulation of autophagy. Here we showed that cytosolic electron-dense particles appear in the cytoplasm of C2C12 myoblasts in which PARP2 is silenced by shRNA. The cytosolic electron-dense bodies resemble autophagic vesicles and, in line with that, we observed an increased number of LC3-positive and Lysotracker-stained vesicles. Silencing of PARP2 did not influence the maximal number of LC3-positive vesicles seen upon chloroquine treatment or serum starvation, suggesting that the absence of PARP2 inhibits autophagic breakdown. Silencing of PARP2 inhibited the activity of AMP-activated kinase (AMPK) and the mammalian target of rapamycin complex 2 (mTORC2). Treatment of PARP2-silenced C2C12 cells with AICAR, an AMPK activator, nicotinamide-riboside (an NAD+ precursor), or EX-527 (a SIRT1 inhibitor) decreased the number of LC3-positive vesicles cells to similar levels as in control (scPARP2) cells, suggesting that these pathways inhibit autophagic flux upon PARP2 silencing. We observed a similar increase in the number of LC3 vesicles in primary PARP2 knockout murine embryonic fibroblasts. We provided evidence that the enzymatic activity of PARP2 is important in regulating autophagy. Finally, we showed that the silencing of PARP2 induces myoblast differentiation. Taken together, PARP2 is a positive regulator of autophagic breakdown in mammalian transformed cells and its absence blocks the progression of autophagy.

Store-operated Ca2+ entry (SOCE) is a Ca2+-entry process activated by the depletion of intracellular stores and has an important role in many cell types. In skeletal muscle, however, its role during physiological muscle activation has been controversial. To address this question, sarcoplasmic reticulum (SR) calcium release in a mouse strain with a naturally occurring mutation in the myostatin gene (Compact (Cmpt)) leading to a hypermuscular yet reduced muscle-force phenotype was compared to that in wild-type mice. To elicit Ca2+ release from the SR of flexor digitorum brevis (FDB) fibers, either a ryanodine receptor agonist (4-chloro-meta-cresol) or depolarizing pulses were used. In muscles from Cmpt mice, endogenous protein levels of STIM1 and Orai1 were reduced, and consequently, SOCE after 4-chloro-meta-cresol-induced store depletion was suppressed. Although the voltage dependence of SR calcium release was not statistically different between wild-type and Cmpt fibers, the amount of releasable calcium was significantly reduced in the latter, indicating a smaller SR content. To assess the immediate role of SOCE in replenishing the SR calcium store, the evolution of intracellular calcium concentration during a train of long-lasting depolarizations to a maximally activating voltage was monitored. Cmpt mice exhibited a faster decline in calcium release, suggesting a compromised ability to refill the SR. A simple model that incorporates a reduced SOCE as an important partner in regulating immediate calcium influx through the surface membrane readily accounts for the steady-state reduction in SR calcium content and its more pronounced decline after calcium release.

Background All known biological functions of the pro-inflammatory cytokine interleukin-1β (IL-1β) are mediated by type 1 interleukin receptor (IL-1R1). IL-1β–IL-1R1 signaling modulates various neuronal functions including spinal pain processing. Although the role of IL-1β in pain processing is generally accepted, there is a discussion in the literature whether IL-1β exerts its effect on spinal pain processing by activating neuronal or glial IL-1R1. To contribute to this debate, here we investigated the expression and cellular distribution of IL-1R1 in the superficial spinal dorsal horn in control animals and also in inflammatory pain. Methods Experiments were performed on rats and wild type as well as IL-1R1-deficient mice. Inflammatory pain was evoked by unilateral intraplantar injection of complete Freund adjuvant (CFA). The nociceptive responsiveness of control and CFA-treated animals were tested daily for withdrawal responses to mechanical and thermal stimuli before and after CFA injection. Changes in the expression of 48 selected genes/mRNAs and in the quantity of IL-1R1 protein during the first 3 days after CFA injection were measured with the TaqMan low-density array method and Western blot analysis, respectively. The cellular localization of IL-1R1 protein was investigated with single and double staining immunocytochemical methods. Results We found a six times and two times increase in IL-1R1 mRNA and protein levels, respectively, in the dorsal horn of CFA-injected animals 3 days after CFA injection, at the time of the summit of mechanical and thermal allodynia. Studying the cellular distribution of IL-1R1, we found an abundant expression of IL-1R1 on the somatodendritic compartment of neurons and an enrichment of the receptor in the postsynaptic membranes of some excitatory synapses. In contrast to the robust neuronal localization, we observed only a moderate expression of IL-1R1 on astrocytes and a negligible one on microglial cells. CFA injection into the hind paw caused a remarkable increase in the expression of IL-1R1 in neurons, but did not alter the glial expression of the receptor. Conclusion The results suggest that IL-1β exerts its effect on spinal pain processing primarily through neuronal IL-1R1, but it can also interact in some extent with IL-1R1 expressed by astrocytes.

Keratinocytes provide the first line of defense of the human body against carcinogenic ultraviolet (UV) radiation. Acute and chronic UVB-mediated cellular responses were widely studied. However, little is known about the role of mitochondrial regulation in UVB-induced DNA damage. Here, we show that poly (ADP-ribose) polymerase 1 (PARP1) and ataxia-telangiectasia-mutated (ATM) kinase, two tumor suppressors, are important regulators in mitochondrial alterations induced by UVB. Our study demonstrates that PARP inhibition by ABT-888 upon UVB treatment exacerbated cyclobutane pyrimidine dimers (CPD) accumulation, cell cycle block and cell death and reduced cell proliferation in premalignant skin keratinocytes. Furthermore, in human keratinocytes UVB enhanced oxidative phosphorylation (OXPHOS) and autophagy which were further induced upon PARP inhibition. Immunoblot analysis showed that these cellular responses to PARP inhibition upon UVB irradiation strongly alter the phosphorylation level of ATM, adenosine monophosphate-activated kinase (AMPK), p53, protein kinase B (AKT), and mammalian target of rapamycin (mTOR) proteins. Furthermore, chemical inhibition of ATM led to significant reduction in AMPK, p53, AKT, and mTOR activation suggesting the central role of ATM in the UVB-mediated mitochondrial changes. Our results suggest a possible link between UVB-induced DNA damage and metabolic adaptations of mitochondria and reveal the OXPHOS-regulating role of autophagy which is dependent on key metabolic and DNA damage regulators downstream of PARP1 and ATM.

Poly(ADP-ribose) polymerase (PARP)10 is a PARP family member that performs mono-ADP-ribosylation of target proteins. Recent studies have linked PARP10 to metabolic processes and metabolic regulators that prompted us to assess whether PARP10 influences mitochondrial oxidative metabolism. The depletion of PARP10 by specific shRNAs increased mitochondrial oxidative capacity in cellular models of breast, cervical, colorectal and exocrine pancreas cancer. Upon silencing of PARP10, mitochondrial superoxide production decreased in line with increased expression of antioxidant genes pointing out lower oxidative stress upon PARP10 silencing. Improved mitochondrial oxidative capacity coincided with increased AMPK activation. The silencing of PARP10 in MCF7 and CaCo2 cells decreased the proliferation rate that correlated with increased expression of anti-Warburg enzymes (Foxo1, PGC-1α, IDH2 and fumarase). By analyzing an online database we showed that lower PARP10 expression increases survival in gastric cancer. Furthermore, PARP10 expression decreased upon fasting, a condition that is characterized by increases in mitochondrial biogenesis. Finally, lower PARP10 expression is associated with increased fatty acid oxidation.