Neurosteroid pregnenolone sulfate - revealing of its site of action at the N-methyl-D-aspartate receptor (26.10. 2020)

NMDA receptors are ion channels involved in the transmission of electrical signals between nerve cells. Their insufficient function contributes to severe neuropsychiatric disorders such as schizophrenia or autism. NMDA receptor activity can be increased by a variety of compounds, including neurosteroids. In collaboration with the Institute of Organic Chemistry and Biochemistry CAS, we identified the binding site and elucidated the mechanism of action of the naturally occurring neurosteroid pregnenolone sulfate (PES). PES binds to the interface of membrane domains of the NMDA receptor in the closed state, and subsequent activation of the receptor by glutamate leads to a rearrangement of the membrane domains and stabilization of the open state of the NMDA receptor ion channel. These findings may contribute to the development of new drugs for the treatment of diseases associated with the dysfunction of the glutamatergic system.

The interaction of the PES molecule (green) with the membrane domains of the NMDA receptor in the open state was obtained by docking followed by molecular dynamics simulation. Amino acids involved in the interaction with the PES molecule are highlighted in red.

Hrčka Krausová B, Kysilov B, Černý J, Vyklický V, Smejkalová T, Ladislav M, Balík A, Kořínek M, Chodounská H, Kudová E, Vyklický ml. L. Site of Action of Brain Neurosteroid Pregnenolone Sulfate at the N-Methyl-D-Aspartate Receptor. Journal of Neuroscience. 2020; 40 (31) 5922-5936. IF: 5.673 

Dietary supplementation with Krill oil as a novel strategy to improve insulin sensitivity and reduce liver fat accumulation in obesity (20.10. 2020)

Lifestyle interventions including healthy nutrition represent an essential part of prevention and treatment strategies for metabolic sequelae of obesity. Krill oil as an extract from the Antarctic krill Euphausia superba (i.e. crustaceans found in Antarctic waters) is a relatively new source of omega-3 polyunsaturated fatty acids (Omega-3). It has been shown that dietary supplementation with Omega-3, usually as part of a triacylglycerol- or ethyl ester-based concentrate, may reduce inflammation and excessive amounts of fat in the liver (i.e. hepatic steatosis).

However, in obese type 2 diabetic patients, the use of Omega-3 may not necessarily improve insulin sensitivity and may impair long-term control of glucose metabolism. Therefore, we tested in obese mice whether supplementation with Omega-3 using an alternative lipid carrier, i.e. phospholipids from Kril oil, could have beneficial metabolic effects while also improving glucose metabolism and insulin sensitivity.

Our studies showed that Krill oil supplementation was able to reduce hepatic steatosis to a greater extent than Omega-3 given as triacylglycerols, and this effect of Krill oil was associated with improved insulin sensitivity in the liver and at the whole-body level. The beneficial effect of Krill oil on glucose homeostasis was associated not only with the improved bioavailability of Omega-3 (e.g. EPA, n-3 DPA) in tissues, but also with circulating levels of palmitoleic acid, a previously identified lipokine (i.e. hormone of a lipid nature) with insulin-sensitising effects whose content in Krill oil is incerased. In addition, Krill oil more effectively then Omega-3 triacylglycerols induced catabolism of fatty acids from the diet directly in the intestine, which could contribute to its strong antisteatotic effects in the liver. Our findings provide a general rationale for using Omega-3-containing phospholipids as nutritional supplements with potent insulin-sensitizing and antisteatotic effects.

Male C57BL/6N mice were fed for 8 weeks a corn oil-based high-fat diet (cHF) alone or supplemented with Omega-3 contained in triacylglycerols (cHF+ω3TG) or Krill oil phospholipids (cHF+ω3PL); lean controls were fed a low-fat standard chow (A). Administration of cHF worsened insulin sensitivity (determined by measuring the glucose infusion rate during the hyperinsulinemic-euglycemic clamp; B – upper part) and caused significant accumulation of lipids in the liver (i.e. hepatic steatosis; B – lower part). While insulin sensitivity was almost preserved and hepatic steatosis virtually eliminated in cHF+ω3PL mice, much less pronounced effects were observed in cHF+ω3TG mice. Favorable metabolic changes observed after Krill oil administration may be related to increased circulating levels of insulin-sensitizing lipokine palmitoleate (POA; C – upper part) and increased fatty acid catabolism directly in the intestinal tissue (POA; C – lower part).

Rossmeisl M, Pavlisova J, Bardova K, Kalendova V, Buresova J, Kuda O, Kroupova P, Stankova B, Tvrzicka E, Fiserova E, Horakova O, Kopecky J. Increased plasma levels of palmitoleic acid may contribute to beneficial effects of Krill oil on glucose homeostasis in dietary obese mice. Biochim Biophys Acta Mol Cell Biol Lipids. 2020;1865(8):158732. IF: 4.519

Kroupova P, van Schothorst EM, Keijer J, Bunschoten A, Vodicka M, Irodenko I, Oseeva M, Zacek P, Kopecky J, Rossmeisl M, Horakova O. Omega-3 Phospholipids from Krill Oil Enhance Intestinal Fatty Acid Oxidation More Effectively than Omega-3 Triacylglycerols in High-Fat Diet-Fed Obese Mice. Nutrients. 2020;12(7):2037. IF: 4.546

Novel mechanism of insulin secretion highlights the importance of redox signalization (14.10. 2020)

Insulin secretion by β cells of pancreatic islets is essential for the maintenance of body glucose homeostasis. Its disturbance leads to the development of diabetes. Till now the mechanism of secretion has been linked exclusively to increased concentration of cellular energy molecule ATP  initiating the cascade of insulin secretion. Our results showed that besides ATP the insulin secretion requires redox signalization. Specifically, we point out NADPH oxidase, isoform 4, which activity increases upon glucose induction. This leads to transient increase of pro-oxidative molecule, hydrogen peroxide, which together with ATP facilitate insulin secretion. Our results emphasize the essential role of redox signalization in the physiology of β cell.

Plecitá-Hlavatá L, Jabůrek M, Holendová B, Tauber J, Pavluch V, Berková Z,  Cahová M,  Schröder K, Brandes R.P, Siemen D, Ježek P Glucose-Stimulated Insulin Secretion Fundamentally Requires H(2)O(2)Signaling by NADPH Oxidase 4 . Diabetes. 2020; 69(7); 1341-1354,IF: 7.720

Detailed characterization of redox status in β cells contributes to the understanding of insulin secretion mechanism

We described the significance of redox signalization in insulin secretion process as the response to an increased level of body glucose in the publication above. The essential enzyme participating in this mechanism is cytosolic NADPH oxidase. It is known, that mitochondria produce superoxide and other reactive oxygen species (ROS) besides energy molecule ATP.  Mitochondria derived ROS can potentially be involved in overall cellular redox state in β cell and thus can impact insulin secretion. We have shown in this paper that glucose induction unexpectedly reduces superoxide production in mitochondria and we attempted to describe the molecular mechanism of such decrease.  For this study, we used novel probes and method of fluorescent microscopy enabling to follow redox status changes in real-time and in various cellular compartments.

(A) Novel molecular mechanism of insulin secretion emphasizing the role of redox signalization in β cells induced by various substrates (glucose, fatty acids (FA), amino acids (AA), Arginin (Arg)).

(B, C) Overview of major metabolic and redox fluxes in β cell under low, non-stimulating, glucose levels (B, causing no insulin secretion) and under high glucose levels inducing insulin secretion (C).

Plecitá-Hlavatá L, Engstová H, Holendová B, Tauber J, Špaček T, Petrásková L, Křen V, Špačková J, Gotvaldová K,Ježek J, Dlasková A, Smolková K, Ježek P Mitochondrial Superoxide Production Decreases on Glucose-Stimulated Insulin Secretion in Pancreatic beta Cells Due to Decreasing Mitochondrial Matrix NADH/NAD(+) Ratio . Antioxidants & Redox Signaling. 2020; 33(12); 789-815 . IF: 6.323

Losartan attenuates neuroinflammation and neuropathic pain in paclitaxel-induced peripheral neuropathy (13.10. 2020)

Paclitaxel-induced peripheral neuropathy in patients is often associated with neuropathic pain and neuroinflammation in the central and peripheral nervous system. The analgesic treatments available for this condition have numerous serious side effects and their efficacy is low. There is a great need to understand the neuropathic pain mechanisms involved in order to find new analgesic drugs. Frequently used antihypertensive drug Losartan, an angiotensin II receptor type 1 (AT1R) blocker, was shown to have some anti-inflammatory and neuroprotective effects in different disease models. In our work we have shown that systemic Losartan treatment attenuated significantly mechanical allodynia in a rodent model of Paclitaxel-induced neuropathic pain. Losartan also significantly reduced paclitaxel-induced neuroinflammatory changes and induced expression of pro-resolving markers (Arginase 1 and IL-10) indicating a possible shift in macrophage polarization. Considering the good safety profile of Losartan, it may be considered as a possible novel treatment strategy for patients with paclitaxel induced neuropathic pain.

Kalynovska N, Diallo M, Sotáková-Kašparová D, Paleček J Losartan attenuates neuroinflammation and neuropathic pain in paclitaxel-induced peripheral neuropathy. Journal of Cellular and Molecular Medicine 24, 14 (2020), 7949-7958. IF: 4.486

Signalling bias as the way to side-effect-free medication (22.7. 2020)

More than 30 % of currently marketed medications act via G-protein coupled receptors (GPCRs). This type of membrane receptors is involved in the control of a wide range of physiological processes, from the processing of sensory stimuli, through the regulation of behavior and mood, hormonal and immune responses, to the control of autonomic functions and cell proliferation. Thus, GPCRs represent one of the most important pharmacotherapeutic targets. In contrast to traditional agonists activating multiple signalling pathways, agonists biased towards a given signalling pathway represent a new generation of drugs with increased specificity and fewer adverse effects. Enormous research has been done on agonists biased towards either G-protein or arrestin mediated pathway.

In our laboratory we focus on the study of muscarinic acetylcholine receptors, which belong to the typical representatives of GPCR. Here, as a proof of concept, we demonstrate unprecedented signalling bias solely at the level of G‑protein-mediated signalling. We present agonists of muscarinic acetylcholine receptors, a member of GPCR family, that exclusively inhibit cAMP synthesis through activation of Gi‑protein pathway and thus are functionally selective for M2 and M4 receptor subtypes. Muscarinic receptors M2, M4 represent one of the pharmacological targets in the treatment of pain. Newly discovered agonists may lead to the development of new non-addictive analgesics such as opiates, or immunity-weakening as steroid analgesics. Such muscarinic analgesics would not cause side effects mediated by activation of the Gq protein signaling pathway, such as incontinence, excessive salivation and sweating, and more.

A) General scheme of coupling GPCRs with individual subtypes of G‑proteins or arrestins after activation by a traditional full agonist (left) and an agonist preferring Gi‑protein activation (right). AC, adenylyl cyclase; PLC, phospholipase C. B) Activation of M2 receptor by the traditional agonist (blue) resulting in stimulation of the Gi and Gs‑protein pathway leading to a decrease in cAMP level in the cell at low concentration of agonist and an increase in cAMP level at high concentrations of the agonist. Activation of M2 receptor by two different Gi‑biased agonists (red, orange) resulting in stimulation of the Gi‑protein pathway only and decrease in cAMP level.

Randáková A, Nelic D, Ungerová D, Nwokoye P, Su Q, Doležal V, El-Fakahany EE, Boulos J, Jakubík J. Novel M2-Selective, Gi-Biased Agonists of Muscarinic Acetylcholine Receptors. British Journal of Pharmacology. 2020; 177(9); 2073-2089 . IF: 7.730