scholarly journals Reducing hepatic PKD activity lowers circulating VLDL cholesterol

2020 ◽  
Vol 246 (3) ◽  
pp. 265-276 ◽  
Author(s):  
Amanda J Genders ◽  
Timothy Connor ◽  
Shona Morrison ◽  
Simon T Bond ◽  
Brian G Drew ◽  
...  
Keyword(s):  
Glucose Homeostasis ◽  
Dominant Negative ◽  
Whole Body ◽  
Protein Kinase D ◽  
Adeno Associated Virus ◽  
Vldl Cholesterol ◽  
Insulin Tolerance ◽  
Fatty Acid Accumulation ◽  
Fasting And Refeeding

Protein kinase D (PKD) is emerging as an important kinase regulating energy balance and glucose metabolism; however, whether hepatic PKD activity can be targeted to regulate these processes is currently unclear. In this study, hepatic PKD activity was reduced using adeno-associated virus vectors to express a dominant-negative (DN) version of PKD1, which impairs the action of all three PKD isoforms. In chow-fed mice, hepatic DN PKD expression increased whole-body glucose oxidation, but had only mild effects on glucose and insulin tolerance and no effects on glucose homeostasis following fasting and refeeding. However, circulating VLDL cholesterol was reduced under these conditions and was associated with hepatic fatty acid accumulation, but not lipids involved in lipoprotein synthesis. The limited effects on glucose homeostasis in DN PKD mice was despite reduced expression of gluconeogenic genes under both fasted and refed conditions, and enhanced pyruvate tolerance. The requirement for PKD for gluconeogenic capacity was supported by in vitro studies in cultured FAO hepatoma cells expressing DN PKD, which produced less glucose under basal conditions. Although these pathways are increased in obesity, the expression of DN PKD in the liver of mice fed a high-fat diet had no impact on glucose tolerance, insulin action, pyruvate tolerance or plasma VLDL. Together, these data suggest that PKD signalling in the liver regulates metabolic pathways involved in substrate redistribution under conditions of normal nutrient availability, but not under conditions of overnutrition such as in obesity.

scholarly journals p-Coumaric Acid Enhances Hypothalamic Leptin Signaling and Glucose Homeostasis in Mice via Differential Effects on AMPK Activation

2021 ◽  
Vol 22 (3) ◽  
pp. 1431
Author(s):  
Linh V. Nguyen ◽  
Khoa D. A. Nguyen ◽  
Chi-Thanh Ma ◽  
Quoc-Thai Nguyen ◽  
Huong T. H. Nguyen ◽  
...  
Keyword(s):  
Glucose Homeostasis ◽  
Blood Glucose Control ◽  
Whole Body ◽  
Ampk Activation ◽  
Coumaric Acid ◽  
Leptin Signaling ◽  
Differential Effects ◽  
Leptin Sensitivity ◽  
Ampk Activity

AMP-activated protein kinase (AMPK) plays a crucial role in the regulation of energy homeostasis in both peripheral metabolic organs and the central nervous system. Recent studies indicated that p-Coumaric acid (CA), a hydroxycinnamic phenolic acid, potentially activated the peripheral AMPK pathway to exert beneficial effects on glucose metabolism in vitro. However, CA’s actions on central AMPK activity and whole-body glucose homeostasis have not yet been investigated. Here, we reported that CA exhibited different effects on peripheral and central AMPK activation both in vitro and in vivo. Specifically, while CA treatment promoted hepatic AMPK activation, it showed an inhibitory effect on hypothalamic AMPK activity possibly by activating the S6 kinase. Furthermore, CA treatment enhanced hypothalamic leptin sensitivity, resulting in increased proopiomelanocortin (POMC) expression, decreased agouti-related peptide (AgRP) expression, and reduced daily food intake. Overall, CA treatment improved blood glucose control, glucose tolerance, and insulin sensitivity. Together, these results suggested that CA treatment enhanced hypothalamic leptin signaling and whole-body glucose homeostasis, possibly via its differential effects on AMPK activation.

scholarly journals Protein kinase D inhibits plasma membrane Na+/H+exchanger activity

1999 ◽  
Vol 277 (6) ◽  
pp. C1202-C1209 ◽  
Author(s):  
Robert S. Haworth ◽  
James Sinnett-Smith ◽  
Enrique Rozengurt ◽  
Metin Avkiran
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Protein Kinase D ◽  
Wild Type ◽  
Ph Indicator

The regulation of plasma membrane Na+/H+exchanger (NHE) activity by protein kinase D (PKD), a novel protein kinase C- and phorbol ester-regulated kinase, was investigated. To determine the effect of PKD on NHE activity in vivo, intracellular pH (pHi) measurements were made in COS-7 cells by microepifluorescence using the pH indicator cSNARF-1. Cells were transfected with empty vector (control), wild-type PKD, or its kinase-deficient mutant PKD-K618M, together with green fluorescent protein (GFP). NHE activity, as reflected by the rate of acid efflux ( J H), was determined in single GFP-positive cells following intracellular acidification. Overexpression of wild-type PKD had no significant effect on J H(3.48 ± 0.25 vs. 3.78 ± 0.24 mM/min in control at pHi 7.0). In contrast, overexpression of PKD-K618M increased J H (5.31 ± 0.57 mM/min at pHi 7.0; P < 0.05 vs. control). Transfection with these constructs produced similar effects also in A-10 cells, indicating that native PKD may have an inhibitory effect on NHE in both cell types, which is relieved by a dominant-negative action of PKD-K618M. Exposure of COS-7 cells to phorbol ester significantly increased J H in control cells but failed to do so in cells overexpressing either wild-type PKD (due to inhibition by the overexpressed PKD) or PKD-K618M (because basal J Hwas already near maximal). A fusion protein containing the cytosolic regulatory domain (amino acids 637–815) of NHE1 (the ubiquitous NHE isoform) was phosphorylated in vitro by wild-type PKD, but with low stoichiometry. These data suggest that PKD inhibits NHE activity, probably through an indirect mechanism, and represents a novel pathway in the regulation of the exchanger.

scholarly journals A Hierarchical Whole-body Modeling Approach Elucidates the Link between in Vitro Insulin Signaling and in Vivo Glucose Homeostasis

2011 ◽  
Vol 286 (29) ◽  
pp. 26028-26041 ◽  
Author(s):  
Elin Nyman ◽  
Cecilia Brännmark ◽  
Robert Palmér ◽  
Jan Brugård ◽  
Fredrik H. Nyström ◽  
...  
Keyword(s):  
Glucose Homeostasis ◽  
Insulin Signaling ◽  
Whole Body ◽  
Modeling Approach

Prolonged β2-adrenergic agonist treatment improves glucose homeostasis in diet-induced obese UCP1-/- mice.

2021 ◽  
Author(s):  
Sten van Beek ◽  
Anastasia Kalinovich ◽  
Gert Schaart ◽  
Tore Bengtsson ◽  
Joris Hoeks
Keyword(s):  
Oxygen Consumption ◽  
Glucose Homeostasis ◽  
Uncoupling Protein ◽  
Fasting Blood Glucose ◽  
Whole Body ◽  
Oral Glucose ◽  
High Fat ◽  
Insulin Tolerance ◽  
Brown Adipose ◽  
Fat Feeding

Objectives- Prolonged supplementation with the β2-agonist clenbuterol improves glucose homeostasis in diabetic rodents, likely via β2-adrenoceptor (β2-AR)-mediated effects in the skeletal muscle and liver. However, since rodents have, in contrast to- especially diabetic- humans, substantial quantities of brown adipose tissue (BAT) and clenbuterol has affinity to β1- and β3-ARs, the contribution of BAT to these improvements is unclear. Therefore, we investigated clenbuterol-mediated improvements in glucose homeostasis in uncoupling protein 1 deficient (UCP1-/-) mice, lacking thermogenic BAT, vs. wild-type (WT) mice. Methods- Anaesthetized WT and UCP1-/- C57Bl/6 mice were injected with saline or clenbuterol and whole-body oxygen consumption was measured. Furthermore, male WT and UCP1-/- C57Bl/6 mice were subjected to 17-weeks of chow feeding, high-fat feeding or high-fat feeding with clenbuterol treatment between week 13-17. Body composition was measured weekly with MRI. Oral glucose tolerance and insulin tolerance tests were performed in week 15 and 17, respectively. Results- Clenbuterol increased oxygen consumption ~2-fold in WT mice. This increase was blunted in UCP1-/- mice, indicating clenbuterol-mediated activation of BAT thermogenesis. High-fat feeding induced diabetogenic phenotypes in both genotypes. However, 2-weeks clenbuterol treatment significantly reduced fasting blood glucose by 12.9% in WT and 14.8% in UCP1-/- mice. Clenbuterol treatment improved glucose and insulin tolerance in both genotypes compared to HFD controls and normalized to chow-fed control mice independent of body mass and -composition alterations. Conclusions- Clenbuterol improved whole-body glucose homeostasis independent of UCP1. Given the low human abundancy of BAT, β2-agonist treatment provides a potential novel route for glucose disposal in diabetic humans.

Abstract 196: Genetically Modified Human Mesenchymal Stromal Cells (MSCs) Help Improve Glucose Homeostasis by Reducing Inflammation and Promotes Browning of Visceral Fat

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Sabyasachi Sen ◽  
Cleyton Domingues ◽  
Nabanita Kundu ◽  
Neeki Ahmadi
Keyword(s):  
Mrna Expression ◽  
Glucose Homeostasis ◽  
Visceral Fat ◽  
Inflammatory Markers ◽  
Tolerance Test ◽  
Mice Model ◽  
Fat Depots ◽  
Insulin Tolerance ◽  
Omental Fat

Background: MSCs are multipotent cells that can home-in to a site of inflammation. Upregulation of specific antioxidants in MSCs reduces intracellular inflammation and ROS formation in a hyperglycemic condition. Hypothesis: Antioxidant over-expressed MSCs will reach fat depots, reduce local & systemic inflammation and improve glucose homeostasis in diet-induced obese (DIO, 60% and 45% fat diet) hyperglycemic mouse models. Methods: We used GFP-containing adenoviral constructs to upregulate intracellular (SOD1, SOD2, Catalase) and extracellular (SOD3) antioxidants in human adipose-derived MSCs. The modified MSCs were delivered in DIO mice. Results: In-vitro, SOD2 (mitochondrial anti-oxidant) upregulation showed reduced inflammatory markers IL6 and TNFa mRNA while PCG1A mRNA (a gene upstream of UCP1), upregulated. SOD2 upregulated MSC delivery in both DIO models demonstrated improved glucose tolerance test (GTT) at week 4 compared to SOD1-MSC and Null-MSC (control). Catalase-MSC delivery not only improved GTT but also improved insulin tolerance test (ITT) in 60% DIO mice. Interestingly, RT-PCR of pericardial fat showed significant increases in mRNA expression of both UCP1 (25-100,000-fold) and PRDM-16 (2-10-fold) in both DIO mice models that received antioxidants upregulated MSCs, compared to mice receiving Null MSCs. For omental fat, an increase in mRNA expression of UCP1 was observed in 60% fat DIO mice (1,000-6000 fold) for SODs 1-3 and catalase, while for 45% DIO mice only those receiving SOD1 & SOD2 upregulated MSCs presented UCP1 mRNA upregulation (1,000 to 11,000-fold). Omental Fat histology showed less hyperplastic fat with SOD2 and Catalase-MSCs. UCP1 staining of omental fat was also positive with SOD2-MSC. Inflammatory molecules such as IL-6 and TNF alpha levels by ELISA, were reduced with SOD2-MSC in 60% DIO mice model. Conclusion: We conclude that delivery of antioxidant upregulated MSCs to the inflamed adipocyte depots in diabetic DIO models appear to upregulate UCP1 and PRDM-16 in visceral fat while reducing systemic inflammatory markers, which may explain improvements noted in GTT and ITT. Delivery of modified MSC is a novel & robust therapeutic tool that improves glucose homeostasis in diet induced diabetes.

scholarly journals miR-200a Attenuated Doxorubicin-Induced Cardiotoxicity through Upregulation of Nrf2 in Mice

2019 ◽  
Vol 2019 ◽  
pp. 1-13 ◽  
Author(s):  
Xiaoping Hu ◽  
Huagang Liu ◽  
Zhiwei Wang ◽  
Zhipeng Hu ◽  
Luocheng Li
Keyword(s):  
Troponin I ◽  
Contractile Function ◽  
Cell Loss ◽  
Whole Body ◽  
Inflammatory Factors ◽  
Adeno Associated Virus ◽  
Nrf2 Activation ◽  
Nrf2 Signaling Pathway ◽  
Body Wasting

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) was closely involved in doxorubicin- (DOX-) induced cardiotoxicity. MicroRNA-200a (miR-200a) could target Keap1 mRNA and promote degradation of Keap1 mRNA, resulting in Nrf2 activation. However, the role of miR-200a in DOX-related cardiotoxicity remained unclear. Our study is aimed at investigating the effect of miR-200a on DOX-induced cardiotoxicity in mice. For cardiotropic expression, male mice received an injection of an adeno-associated virus 9 (AAV9) system carrying miR-200a or miR-scramble. Four weeks later, mice received a single intraperitoneal injection of DOX at 15 mg/kg. In our study, we found that miR-200a mRNA was the only microRNA that was significantly decreased in DOX-treated mice and H9c2 cells. miR-200a supplementation blocked whole-body wasting and heart atrophy caused by acute DOX injection, decreased the levels of cardiac troponin I and the N-terminal probrain natriuretic peptide, and improved cardiac and adult cardiomyocyte contractile function. Moreover, miR-200a reduced oxidative stress and cardiac apoptosis without affecting matrix metalloproteinase and inflammatory factors in mice with acute DOX injection. miR-200a also attenuated DOX-induced oxidative injury and cell loss in vitro. As expected, we found that miR-200a activated Nrf2 and Nrf2 deficiency abolished the protection provided by miR-200a supplementation in mice. miR-200a also provided cardiac benefits in a chronic model of DOX-induced cardiotoxicity. In conclusion, miR-200a protected against DOX-induced cardiotoxicity via activation of the Nrf2 signaling pathway. Our data suggest that miR-200a may represent a new cardioprotective strategy against DOX-induced cardiotoxicity.

scholarly journals Galectin-3 gene deletion results in defective adipose tissue maturation and impaired insulin sensitivity and glucose homeostasis

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Claudia Blasetti Fantauzzi ◽  
Carla Iacobini ◽  
Stefano Menini ◽  
Martina Vitale ◽  
Gian Pio Sorice ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Insulin Sensitivity ◽  
Glucose Homeostasis ◽  
Cell Function ◽  
Whole Body ◽  
Metabolic Derangement ◽  
Abnormal Glucose Regulation ◽  
Galectin 3 ◽  
Β Cell Function

AbstractAdiposopathy is a pathological adipose tissue (AT) response to overfeeding characterized by reduced AT expandability due to impaired adipogenesis, which favors inflammation, insulin resistance (IR), and abnormal glucose regulation. However, it is unclear whether defective adipogenesis causes metabolic derangement also independently of an increased demand for fat storage. As galectin-3 has been implicated in both adipocyte differentiation and glucose homeostasis, we tested this hypothesis in galectin-3 knockout (Lgal3−/−) mice fed a standard chow. In vitro, Lgal3−/− adipocyte precursors showed impaired terminal differentiation (maturation). Two-month-old Lgal3−/− mice showed impaired AT maturation, with reduced adipocyte size and expression of adipogenic genes, but unchanged fat mass and no sign of adipocyte degeneration/death or ectopic fat accumulation. AT immaturity was associated with AT and whole-body inflammation and IR, glucose intolerance, and hyperglycemia. Five-month-old Lgal3−/− mice exhibited a more mature AT phenotype, with no difference in insulin sensitivity and expression of inflammatory cytokines versus WT animals, though abnormal glucose homeostasis persisted and was associated with reduced β-cell function. These data show that adipogenesis capacity per se affects AT function, insulin sensitivity, and glucose homeostasis independently of increased fat intake, accumulation and redistribution, thus uncovering a direct link between defective adipogenesis, IR and susceptibility to diabetes.

scholarly journals Mathematical Modeling for the Physiological and Clinical Investigation of Glucose Homeostasis and Diabetes

2020 ◽  
Vol 11 ◽  
Author(s):  
Andrea Mari ◽  
Andrea Tura ◽  
Eleonora Grespan ◽  
Roberto Bizzotto
Keyword(s):  
Mathematical Modeling ◽  
Glucose Homeostasis ◽  
Cell Function ◽  
Clinical Investigation ◽  
Experimental Tests ◽  
Whole Body ◽  
Mathematical Representation ◽  
Use Of Models

Mathematical modeling in the field of glucose metabolism has a longstanding tradition. The use of models is motivated by several reasons. Models have been used for calculating parameters of physiological interest from experimental data indirectly, to provide an unambiguous quantitative representation of pathophysiological mechanisms, to determine indices of clinical usefulness from simple experimental tests. With the growing societal impact of type 2 diabetes, which involves the disturbance of the glucose homeostasis system, development and use of models in this area have increased. Following the approaches of physiological and clinical investigation, the focus of the models has spanned from representations of whole body processes to those of cells, i.e., from in vivo to in vitro research. Model-based approaches for linking in vivo to in vitro research have been proposed, as well as multiscale models merging the two areas. The success and impact of models has been variable. Two kinds of models have received remarkable interest: those widely used in clinical applications, e.g., for the assessment of insulin sensitivity and β-cell function and some models representing specific aspects of the glucose homeostasis system, which have become iconic for their efficacy in describing clearly and compactly key physiological processes, such as insulin secretion from the pancreatic β cells. Models are inevitably simplified and approximate representations of a physiological system. Key to their success is an appropriate balance between adherence to reality, comprehensibility, interpretative value and practical usefulness. This has been achieved with a variety of approaches. Although many models concerning the glucose homeostasis system have been proposed, research in this area still needs to address numerous issues and tackle new opportunities. The mathematical representation of the glucose homeostasis processes is only partial, also because some mechanisms are still only partially understood. For in vitro research, mathematical models still need to develop their potential. This review illustrates the problems, approaches and contribution of mathematical modeling to the physiological and clinical investigation of glucose homeostasis and diabetes, focusing on the most relevant and stimulating models.

scholarly journals Paradoxical Glucose-Sensitizing yet Proinflammatory Effects of Acute ASP Administration in Mice

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Alexandre Fisette ◽  
Pegah Poursharifi ◽  
Katerina Oikonomopoulou ◽  
Mercedes N. Munkonda ◽  
Marc Lapointe ◽  
...  
Keyword(s):  
Energy Metabolism ◽  
Whole Body ◽  
Diet Induced Obesity ◽  
Insulin Tolerance ◽  
M1 Macrophage ◽  
Acylation Stimulating Protein ◽  
Inflammatory Processes

Acylation stimulating protein (ASP) is an adipokine derived from the immune complement system, which stimulates fat storage and is typically increased in obesity, type 2 diabetes, and cardiovascular disease. Using a diet-induced obesity (DIO) mouse model, the acute effects of ASP on energy metabolism and inflammatory processesin vivowere evaluated. We hypothesized that ASP would specifically exert proinflammatory effects. C57Bl/6 wild-type mice were put on a high-fat-high-sucrose diet for 12 weeks. Mice were then subjected to both glucose and insulin tolerance tests, each manipulation being preceded by recombinant ASP or vehicle (control) bolus injection. ASP supplementation increased whole-body glucose excursion, and this was accomplished with reduced concomitant insulin levels. However, ASP did not directly alter insulin sensitivity. ASP supplementation induced a proinflammatory phenotype, with higher levels of cytokines including IL-6 and TNF-αin plasma and in adipose tissue, liver, and skeletal muscle mRNA. Additionally, ASP increased M1 macrophage content of these tissues. ASP exerted a direct concentration-dependent role in the migration and M1 activation of cultured macrophages. Altogether, thein vivoandin vitroexperiments demonstrate that ASP plays a role in both energy metabolism and inflammation, with paradoxical whole-body glucose-sensitizing yet proinflammatory effects.

scholarly journals Πρόληψη της ανάπτυξης εξάρτησης από τη μορφίνη έπειτα από υπερέκφραση της πρωτεΐνης RGS9 στον εγκέφαλο

2011 ◽  
Author(s):  
Μαρία-Μάρθα Παπαχατζάκη
Keyword(s):  
Dominant Negative ◽  
Adeno Associated Virus

Μέχρι σήμερα τα οπιοειδή παραμένουν τα φάρμακα με την ισχυρότερη αναλγητική ανταπόκριση σε αρκετές μορφές πόνου. Ένα από τα μεγαλύτερα κλινικά αλλά και κοινωνικά προβλήματα στην αντιμετώπιση του χρόνιου πόνου είναι η ανάπτυξη εξάρτησης από τα οπιοειδή σε ασθενείς που χρειάζονται χρόνια αναλγητική θεραπεία και η εμφάνιση στερητικού συνδρόμου μετά την απότομη διακοπή χορήγησης τους. Πέραν όμως από το πρόβλημα της εξάρτησης στην χρόνια λήψη οπιοειδών, οι ασθενείς παρουσιάζουν και μείωση της αναλγητικής τους ανταπόκρισης, λόγω ανάπτυξης ανοχής στην χορηγούμενη δόση μορφίνης. Η ανοχή οδηγεί σε συνεχή αύξηση της δοσολογίας του φαρμάκου, προκειμένου να επιτευχθεί το αναμενόμενο αναλγητικό αποτέλεσμα, με συνέπεια, την ανάπτυξη εξάρτησης στην ουσία και την εμφάνιση σοβαρών παρενεργειών τόσο στο ΚΝΣ, όσο και σε περιφερικούς ιστούς. Όλα αυτά, καθιστούν την χορήγηση της μορφίνης, καθώς και άλλων οπιοειδών φαρμάκων προβληματική, σε ασθενείς που θα την είχαν ανάγκη για μεγάλο χρονικό διάστημα καθώς και περιορισμένη κυρίως σε κλινικές οντότητες με χαμηλό προσδόκιμο ζωής ή τελικού σταδίου νόσου (πχ καρκινοπαθείς). Οι αλλαγές αυτές της δράσης της μορφίνης στον οργανισμό είναι αποτέλεσμα προσαρμοστικών μεταβολών που συμβαίνουν σε κυτταρικό επίπεδο και σε συγκεκριμένα νευρωνικά υποστρώματα. Οι ακριβείς μηχανισμοί αυτών των μεταβολών δεν είναι απολύτως διευκρινισμένοι και πιθανώς συμπεριλαμβάνουν τροποποιήσεις σε πολλαπλά νευρωνικά δίκτυα και στην έκφραση μεγάλου αριθμού γονιδίων, άρα και πρωτεϊνών. Για τον λόγο αυτό, είναι σημαντική η ταυτοποίηση των πρωτεϊνών που συμμετέχουν στους προσαρμοστικούς μηχανισμούς που οδηγούν στην ανάπτυξη ανοχής και εξάρτησης του οργανισμού στην μορφίνη. Μία ομάδα πρωτεϊνών που συμβάλει στους προσαρμοστικούς μηχανισμούς της χρόνιας χορήγησης μορφίνης στον οργανισμό είναι η οικογένεια των RGS πρωτεϊνών. Οι πρωτεΐνες αυτές ρυθμίζουν την ένταση και διάρκεια της δραστικότητας των G πρωτεϊνών, ρυθμίζοντας έτσι έμμεσα το εύρος σηματοδότησης των υποδοχέων που είναι προσδεδεμένοι με τις G πρωτεΐνες. Οι RGS πρωτεΐνες παίζουν ρόλο σε πολλές χρόνιες ασθένειες, όπως η σχιζοφρένεια, η κατάθλιψη και η εξάρτηση στις ουσίες. Συγκεκριμένα, μια σειρά πρόσφατων μελετών, συνδέει την πρωτεΐνη RGS9-2 με την εξάρτηση. Η RGS9-2, η οποία παρουσιάζει πλούσια έκφραση στο κέντρο εθισμού, ρυθμίζει την διάρκεια ενεργοποίησης της άλφα υπομονάδας των G πρωτεϊνών, μέσω κατάλυσης της επανασύνδεσης των α και βγ υπομονάδων της, άρα και την λήξη σηματοδότησης του υποδοχέα. Πολλαπλές in vitro και in vivo μελέτες έδειξαν ότι η RGS9-2 ρυθμίζει δυναμικά την ανταπόκριση των μ-οπιοειδών υποδοχέων (MOR) και παίζει σημαντικό ρόλο στις δράσεις της μορφίνης ενώ επίσης προάγει την ευαισθητοποίηση στις ανταμοιβικές ιδιότητες των οπιοειδών και ψυχοδιεγερτικών ουσιών (Burns&Wensel 2003, Rahman et al 2003, Zachariou et al 2003, Psifogeorgou et al. 2007& 2010). Πιο συγκεκριμένα, προηγούμενες μελέτες του εργαστηρίου μας σε διαγονιδιακά ζώα, με καθολική απαλοιφή του γονιδίου της RGS9, έδειξαν ότι τα ζώα αυτά εμφανίζουν αυξημένη συμπεριφορική ευαισθησία στην ανταμοιβική δράση της μορφίνης, σε σχέση με τα αγρίου τύπου ζώα, αναλγητική ανταπόκριση σε πολύ χαμηλότερες δόσεις από αυτές που χρειάζονταν τα αγρίου τύπου, καθώς και καθυστέρηση στην ανάπτυξη ανοχής (Zachariou et al 2003). Τα δεδομένα αυτά δείχνουν ότι επέμβαση στη δράση της RGS9-2, επηρεάζει δυναμικά τις δράσεις της μορφίνης στο ΚΝΣ και καθιστούν το μόριο αυτό, σημαντικό φαρμακολογικό στόχο. Στόχος της παρούσας διατριβής είναι να διερευνηθούν οι επιδράσεις της στοχευμένης τροποποίησης των επίπεδων της RGS9-2 σε ειδικούς πυρήνες του εγκεφάλου, όπου εμφανίζει πλούσια κατανομή. Το ραχιαίο ραβδωτό και ιδίως ο επικλινής πυρήνας (κοιλιακό ραβδωτό), αποτελούν τους σημαντικότερους πυρήνες στο παθοφυσιολογικό και φαρμακολογικό μονοπάτι του εθισμού, αφού ρυθμίζουν τις ενισχυτικές και εξαρτησιογόνες δράσεις των οπιοειδών έπειτα απο διέγερση των μ-οπιοειδών υποδοχέων και εμφανίζουν τα υψηλότερα επίπεδα έκφρασης της πρωτείνης στον εγκέφαλο. Πέραν των ανωτέρω περιοχών όμως, αρκετά υψηλά επίπεδα έκφρασης της RGS9-2 πρωτεΐνης έχουν ανιχνευθεί και στα οπίσθια κέρατα του νωτιαίου μυελού, που όπως είναι γνωστό, αποτελούν σημαντικές περιοχές νευροδιαβίβασης των επώδυνων ερεθισμάτων, καθώς και στόχο αρκετών αναλγητικών φαρμάκων όπως τα οπιοειδή. Όλες οι προαναφερόμενες περιοχές εκφράζουν σε υψηλά επίπεδα την RGS9-2. Με στόχο την διερεύνηση του ρόλου της RGS9-2 στις συγκεκριμένες περιοχές του ΚΝΣ, δημιουργήθηκαν γενετικά μοντέλα υπερέκφρασης της. Συγκεκριμένα χρησιμοποιήθηκαν AAV ιϊκά οχήματα αδενοσυσχετιζόμενων ιών (adeno associated virus) τα οποία εγχύθηκαν μέσω στερεοταξικής επέμβασης σε επιλεγμένες περιοχές του εγκεφάλου. Οι ιοί που χρησιμοποιούνται στην συγκεκριμένη διατριβή υπερεκφράζουν την RGS9-2 ή την μεταλλαγμένη μορφή της πρωτεΐνης η οποία έχει dominant negative δράση. Συγκεκριμένα, οι ιοί που κατασκευάστηκαν, φέρουν είτε την RGS9-2 σεσημασμένη με GFP, είτε την μεταλλαγμένη μορφή της RGS9-2 DEPless (η RGS9-2 πρωτεΐνη χωρίς το DEP domain της, το οποίο της προσδίδει την ικανότητα πρόσδεσης της στην κυτταρική μεμβράνη, άρα και την δημιουργία σταθερών συμπλόκων κοντά σ' αυτήν) σεσημασμένη με GFP, με στόχο την υπερέκφραση των πρωτεϊνών αυτών στις περιοχές του εγκεφάλου που διοχετεύεται ο ιός. Προ έναρξης ευρείας χρήσης των AAV οχημάτων σε συμπεριφορικές μελέτες έγινε αρχικά ταυτοποίηση του ποσοστού υπερέκφρασης και της κατανομής του ιού στην περιοχή έγχυσης καθώς και στα νευρικά κύτταρα στα οποία εκφράζεται ο ιός υπερέκφρασης της RGS9-2. Μελέτες που έγιναν σε πρωτογενείς κυτταρικές σειρές, δείχνουν ότι η παρουσία της μεταλλαγμένης RGS9-2 (RGS9-2-DEPless) παίζει dominant negative ρόλο (Psifogeorgou et al. 2007), ανταγωνίζεται δηλαδή την δράση της ενδογενούς RGS9-2 πρωτεΐνης σχηματίζοντας σύμπλοκα με τα σηματοδοτικά της μόρια μακριά από την κυτταρική μεμβράνη - λόγω έλλειψης του DEP τμήματος από την κυτταρική δομή της RGS9-2 πρωτεΐνης (Kassi Psifogeorgiou 2007). Επίσης κατασκευάστηκε ιός ο οποίος έφερε μόνο το γονίδιο της EGFP για την παραγωγή της ομάδας των ποντικών μαρτύρων. Στην συνέχεια μελετήθηκαν οι επιπτώσεις της υπερέκφρασης ή ανταγωνισμού δράσης της RGS9-2 στις συγκεκριμένες περιοχές του εγκεφάλου, στην συμπεριφορά των ζώων μετά από εφάπαξ ή χρόνια χορήγηση μορφίνης, μέσω ευρέως γνωστών συμπεριφορικών παραδειγμάτων αξιολόγησης της εξάρτησης, ανταμοιβής, ανοχής και αναλγησίας στην μορφίνη. Μέσω αυτών διαπιστώθηκε: 1) η σπουδαιότητα της δράσης της RGS9-2 στον επικλινή πυρήνα και στους μηχανισμούς ανάπτυξης εθισμού, αναλγησίας και ανοχής στα οπιοειδή, 2) η σπουδαιότητα της δράσης της RGS9-2 στο ραχιαίο ραβδωτό και στους μηχανισμούς εθισμού, αναλγησίας και ανοχής στα οπιοειδή, 3) η περαιτέρω σχέση των πυρήνων αυτών με τον εθισμό και την αναλγησία, καθώς και 4) ο ρόλος της RGS9-2 στα οπίσθια κέρατα του νωτιαίου μυελού στις αναλγητικές δράσεις της μορφίνης.

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