scholarly journals UDP-glucuronosyltransferase 2B15 (UGT2B15) and UGT2B17 Enzymes Are Major Determinants of the Androgen Response in Prostate Cancer LNCaP Cells

2007 ◽  
Vol 282 (46) ◽  
pp. 33466-33474 ◽  
Author(s):  
Sarah Chouinard ◽  
Olivier Barbier ◽  
Alain Bélanger
Keyword(s):  
Culture Media ◽  
Cancer Cell Line ◽  
Mrna Levels ◽  
Uridine Diphosphate ◽  
Lncap Cells ◽  
Protein Levels ◽  
Prostate Cells ◽  
Target Probe ◽  
Interfering Rna ◽  
The Impact

Uridine diphosphate-glucuronosyltransferase 2 (UGT2)B15 and B17 enzymes conjugate dihydrotestosterone (DHT) and its metabolites androstane-3α, 17β-diol (3α-DIOL) and androsterone (ADT). The presence of UGT2B15/B17 in the epithelial cells of the human prostate has been clearly demonstrated, and significant 3α-DIOL glucuronide and ADT-glucuronide concentrations have been detected in this tissue. The human androgen-dependent cancer cell line, LNCaP, expresses UGT2B15 and -B17 and is also capable of conjugating androgens. To assess the impact of these two genes in the inactivation of androgens in LNCaP cells, their expression was inhibited using RNA interference. The efficient inhibitory effects of a UGT2B15/B17 small interfering RNA (siRNA) probe was established by the 70% reduction of these UGT mRNA levels, which was further confirmed at the protein levels. The glucuronidation of dihydrotestosterone (DHT), 3α-DIOL, and ADT by LNCaP cell homogenates was reduced by more than 75% in UGT2B15/B17 siRNA-transfected LNCaP cells when compared with cells transfected with a non-target probe. In UGT2B15/B17-deficient LNCaP cells, we observe a stronger response to DHT than in control cells, as determined by cell proliferation and expression of eight known androgen-sensitive genes. As expected, the amounts of DHT in cell culture media from control cells were significantly lower than that from UGT2B15/B17 siRNA-treated cells, which was caused by a higher conversion to its corresponding glucuronide derivative. Taken together these data support the idea that UGT2B15 and -B17 are critical enzymes for the local inactivation of androgens and that glucuronidation is a major determinant of androgen action in prostate cells.

scholarly journals DBC1 (Deleted in Breast Cancer 1) modulates the stability and function of the nuclear receptor Rev-erbα

2013 ◽  
Vol 451 (3) ◽  
pp. 453-461 ◽  
Author(s):  
Claudia C. S. Chini ◽  
Carlos Escande ◽  
Veronica Nin ◽  
Eduardo N. Chini
Keyword(s):  
Breast Cancer ◽  
Nuclear Receptor ◽  
Clock Genes ◽  
Mrna Levels ◽  
Protein Levels ◽  
The Stability ◽  
And Function ◽  
Interfering Rna ◽  
In Cells

The nuclear receptor Rev-erbα has been implicated as a major regulator of the circadian clock and integrates circadian rhythm and metabolism. Rev-erbα controls circadian oscillations of several clock genes and Rev-erbα protein degradation is important for maintenance of the circadian oscillations and also for adipocyte differentiation. Elucidating the mechanisms that regulate Rev-erbα stability is essential for our understanding of these processes. In the present paper, we report that the protein DBC1 (Deleted in Breast Cancer 1) is a novel regulator of Rev-erbα. Rev-erbα and DBC1 interact in cells and in vivo, and DBC1 modulates the Rev-erbα repressor function. Depletion of DBC1 by siRNA (small interfering RNA) in cells or in DBC1-KO (knockout) mice produced a marked decrease in Rev-erbα protein levels, but not in mRNA levels. In contrast, DBC1 overexpression significantly enhanced Rev-erbα protein stability by preventing its ubiquitination and degradation. The regulation of Rev-erbα protein levels and function by DBC1 depends on both the N-terminal and C-terminal domains of DBC1. More importantly, in cells depleted of DBC1, there was a dramatic decrease in circadian oscillations of both Rev-erbα and BMAL1. In summary, our data identify DBC1 as an important regulator of the circadian receptor Rev-erbα and proposes that Rev-erbα could be involved in mediating some of the physiological effects of DBC1.

scholarly journals Induction of Oxidative Stress and Apoptosis in the Injured Brain: Potential Relevance to Brain Regeneration in Zebrafish.

2021 ◽  
Author(s):  
Surendra Kumar Anand ◽  
Manas Ranjan Sahu ◽  
Amal Chandra Mondal
Keyword(s):  
Oxidative Stress ◽  
Mrna Levels ◽  
Adult Zebrafish ◽  
Zebrafish Model ◽  
Stab Injury ◽  
Zebrafish Brain ◽  
Protein Levels ◽  
Injured Brain ◽  
Brain Regeneration ◽  
The Impact

Abstract In the recent years, zebrafish, owing to its tremendous adult neurogenic capacity, has emerged as a useful vertebrate model to study brain regeneration. Recent findings suggest a significant role of the BDNF/TrkB signaling as a mediator of brain regeneration following a stab injury in the adult zebrafish brain. Since BDNF has been implicated in a plethora of physiological processes, we hypothesized that these processes are affected in the injured zebrafish brain. In this small study, we examined the indicators of oxidative stress and of apoptosis using biochemical assays, RT-PCR and IHC to reflect upon the impact of stab injury on oxidative stress levels and apoptosis in the injured adult zebafish brain. Our results indicate induction of oxidative stress in the injured adult zebrafish brain. Also, apoptosis was induced in the injured brain as indicated by increased protein levels of cleaved caspase3 as well as enhanced mRNA levels of both pro-apoptotic and anti-apoptotic genes. This knowledge contributes to the overall understanding of adult neurogenesis in the zebrafish model and raises new questions pertaining to the compensatory physiological mechanisms in response to traumatic brain injury in the adult zebrafish brain.

scholarly journals Role of UBC9 in the Regulation of the Adipogenic Program in 3T3-L1 Adipocytes

Endocrinology ◽  
2010 ◽  
Vol 151 (11) ◽  
pp. 5255-5266 ◽  
Author(s):  
Angelo Cignarelli ◽  
Mariangela Melchiorre ◽  
Alessandro Peschechera ◽  
Antonella Conserva ◽  
Lucia Adelaide Renna ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Small Interfering Rna ◽  
Protein Modification ◽  
Covalent Attachment ◽  
Mrna Levels ◽  
Protein Levels ◽  
Mrna Induction ◽  
Induction Of Differentiation ◽  
Interfering Rna

The small ubiquitin-like modifier-conjugating enzyme UBC9, involved in protein modification through covalent attachment of small ubiquitin-like modifier and other less defined mechanisms, has emerged as a key regulator of cell proliferation and differentiation. To explore the role of UBC9 in adipocyte differentiation, the UBC9 protein levels were examined in differentiating 3T3-L1 cells. UBC9 mRNA and protein levels were increased 2.5-fold at d 2 and then gradually declined to basal levels at d 8 of differentiation. In addition, UBC9 was expressed predominantly in the nucleus of preadipocytes but shifted to cytoplasmic compartments after d 4, after induction of differentiation. UBC9 knockdown was then achieved in differentiating 3T3-L1 preadipocytes using a specific small interfering RNA. Oil-Red-O staining demonstrated accumulation of large triglyceride droplets in approximately 90% of control cells, whereas lipid droplets were smaller and evident in only 30% of cells treated with the UBC9-specific small interfering RNA. CCAAT/enhancer-binding protein (C/EBP)-δ, peroxisome proliferator-activated receptor-γ, and C/EBPα mRNA levels were increased severalfold 2–6 d after induction of differentiation in control cells, whereas the expression of these transcription factors was significantly lower in the presence of UBC9 gene silencing. Adenovirus-mediated overexpression of a catalytically inactive mutant UBC9 protein in 3T3-L1 cells resulted in no changes in expression of adipogenic transcription factors and conversion to mature adipocytes as compared with control. In conclusion, UBC9 appears to play an important role in adipogenesis. The temporal profile of UBC9 induction and its ability to affect C/EBPδ mRNA induction support a role for this protein during early adipogenesis.

scholarly journals Role of Rac1 in regulation of NOX5-S function in Barrett's esophageal adenocarcinoma cells

2011 ◽  
Vol 301 (2) ◽  
pp. C413-C420 ◽  
Author(s):  
Jie Hong ◽  
Murray Resnick ◽  
Jose Behar ◽  
Jack Wands ◽  
Ronald A. DeLellis ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Line ◽  
Esophageal Adenocarcinoma ◽  
Endoplasmic Reticulum Membrane ◽  
Mrna Levels ◽  
Protein Levels ◽  
Functional Regulation ◽  
Normal Human ◽  
Species Production ◽  
Interfering Rna

We have shown that a novel NADPH oxidase isoform, NOX5-S, is the major isoform of NADPH oxidases in an esophageal adenocarcinoma (EA) cell line, FLO, and is overexpressed in Barrett's mucosa with high-grade dysplasia. NOX5-S is responsible for acid-induced reactive oxygen species production. In this study, we found that mRNA levels of NOX5-S were significantly higher in FLO EA cells than in the normal human esophageal squamous cell line HET-1A or in a Barrett cell line, BAR-T. The mRNA levels of NOX5-S were also significantly increased in EA tissues. The data suggest that NOX5-S may be important in the development of EA. Mechanisms of functional regulation of NOX5-S are not fully understood. We show that small G protein Rac1 was present in HET-1A cells, BAR-T cells, and EA cell lines FLO and OE33. Rac1 protein levels were significantly higher in FLO and OE33 cells than in HET-1A or BAR-T cells. Knockdown of Rac1 with Rac1 small interfering RNA significantly decreased acid-induced increase in H2O2 production in FLO EA cells. Overexpression of constitutively active Rac1 significantly increased H2O2 production, an increase that was blocked by knockdown of NOX5-S. By immunofluorescence staining and immunoprecipitation, we found that NOX5-S was present in the cytosol of FLO EA cells and colocalized with Rac1 and SERCA1/2 Ca2+-ATPase which is located in the endoplasmic reticulum membrane. We conclude that Rac1 may be important in activation of NOX5-S in FLO EA cells.

scholarly journals Restore the Secretion and Activity of Factor VIII Mutants By Proteostasis Regulator

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1104-1104
Author(s):  
Wei Wei ◽  
Zhigang Liu ◽  
Bin Zhang
Keyword(s):  
Cell Culture ◽  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitor ◽  
Culture Media ◽  
Activity Levels ◽  
Mrna Levels ◽  
Cell Culture Media ◽  
Protein Levels ◽  
Deacetylase Inhibitor ◽  
A2 Domain

Quantitative or qualitative deficiency in factor VIII (FVIII) result in hemophilia A. FVIII deficiency is caused by broad spectrum mutations, and missense mutation is the most common type of mutation, which lead to various defects of FVIII from decreased secretion to impaired function. Cellular protein homeostasis is disturbed by FVIII mutants, and thus excessive misfolding and degradation of FVIII mutant result in FVIII deficiency. We hypothesized that folding and/or trafficking of FVIII can be enhanced by proteostasis regulators, leading to increased coagulation factor levels sufficient to restore hemostasis. We performed a cell-based screening using a FDA-approved drug library and a natural compound library looking for compounds that enhance secretion of FVIII with a B domain deletion. We identified SAHA (Suberanilohydroxamic acid) as a compound that increased both wild-type (WT) and a missense mutant FVIII. SAHA is a histone deacetylase inhibitor that was also reported to enhance proteostaisis of mutant CFTR, a-1 antitrypsin, lysosomal glucocerebrosidase and the GABA receptor. Here we show that SAHA can increase the secretion and activity of FVIII mutants by enhancing their folding and trafficking as well transcription. To confirm the ability of SAHA to enhance FVIII mutant secretion, we first used the WT FVIII A2 domain and the M614T mutant A2 domain to detect the proper working time and concentration of SAHA in HEK 293T cells. M614T is a missense mutation that was shown to cause secretion defect of FVIII in our previous study. Western blot showed a dose-dependent increase in levels of both A2-WT and A2-M614T in cell culture media between 0 and 2.5ug/ml of SAHA after 24-hour treatment. Levels of both WT and mutant A2 domain dropped when SAHA concentration exceeded 5 ug/ml due to cellular toxicity. SAHA enhanced the secretion of A2-M614T much more than that of A2-WT. Of note, protein levels of both A2-WT and A2-M614T in cell lysate were also slightly elevated by SAHA. Time course study showed that the maximum effect on secretion was achieved after 24 hours of SAHA treatment. We next studied the effect of SAHA on activity and secretion of several full-length FVIII mutants (A469S, R527W, R531G, I566T, N582D, S584T, R593C, M614T, R1997G, R2150C), which were shown to decrease FVIII secretion due to various defects in intracellular trafficking in our previous studies. Except for N582D, activity levels of all FVIII mutants in cell culture media were elevated 3-14 folds after a 24-hour SAHA treatment (P<0.05), whereas the activity level of FVIII WT only increased by ~2 folds (P<0.05). Antigen levels of FVIII mutants in both cell lysates and cell culture media increased by ~3 folds following SAHA treatment (P<0.05). The increase in activities of several FVIII mutants (I566T, S584T, R593C, R1997G, R2150C) is higher than the increase in antigen levels of the same mutants in cell culture media, which indicates that, in addition to enhancing secretion, SAHA treatment also increased the proportions of functional proteins in secreted FVIII mutants in cell culture media. To investigate the mechanism, we studied the interactions of WT FVIII and several responsive mutant FVIII (I566T, S584T, R593C and R1997C) with endoplasmic reticulum (ER) chaperones BiP and calreticulin by co-Immunoprecipitation. The protein levels of BiP and calreticulin in HEK 293T cells were not affected by SAHA. However, the interactions of BiP with both WT FVIII and FVIII mutants were remarkably enhanced by SAHA, while no significant differences in the interactions of calreticulin with FVIII WT or FVIII mutants were observed. The effect of SAHA on FVIII secretion can be blocked by simultaneously treating cells with a BiP inhibitor SubAB. We next measured mRNA levels of BiP as well as FVIII in cells expressing FVIII WT and FVIII mutants with or without SAHA treatment via real time PCR. Similar to protein levels, BiP mRNA levels were not changed by SAHA. However, FVIII mRNA levels were elevated by 1.2 to 3.1 folds (P<0.05) by SAHA, in keeping with the histone deacetylase inhibitor activity of SAHA. In summary, we have shown that SAHA increases FVIII antigen and activity levels in cell culture media by increasing FVIII mRNA levels and by enhancing its folding and trafficking via specifically promoting its interaction with BiP. SAHA is a potential proteostaisis regulator that can increase the secretion and restore the function of certain FVIII missense mutants. Disclosures No relevant conflicts of interest to declare.

scholarly journals Mycobacterium tuberculosis Requires Cholesterol Oxidase to Disrupt TLR2 Signalling in Human Macrophages

2019 ◽  
Vol 2019 ◽  
pp. 1-17
Author(s):  
Izabela Szulc-Kielbik ◽  
Michal Kielbik ◽  
Patrycja Przygodzka ◽  
Anna Brzostek ◽  
Jaroslaw Dziadek ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cholesterol Oxidase ◽  
Interleukin 10 ◽  
Signalling Pathway ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Wild Type ◽  
Human Macrophages ◽  
Protein Levels ◽  
The Impact

This study tested the hypothesis that Mycobacterium tuberculosis (Mtb) uses a cholesterol oxidase enzyme (ChoD) to suppress a toll-like receptor type 2- (TLR2-) dependent signalling pathway to modulate macrophages’ immune response. We investigated the impact of Mtb possessing or lacking ChoD as well as TBChoD recombinant protein obtained from Mtb on the expression and activation of two key intracellular proteins involved in TLR2 signalling in human macrophages. Finally, the involvement of TLR2-related signalling proteins in an inflammatory/immunosuppressive response of macrophages to Mtb was evaluated. We demonstrate that wild-type Mtb but not the ∆choD mutant decreased the cytosolic IRAK4 and TRAF6 protein levels while strongly enhancing IRAK4 and TRAF6 mRNA levels in macrophages. Our data show that the TLR2 present on the surface of macrophages are involved in disturbing the signalling pathway by wild-type Mtb. Moreover, recombinant TBChoD effectively decreased the cytosolic level of TRAF6 and lowered the phosphorylation of IRAK4, which strongly confirm an involvement of cholesterol oxidase in affecting the TLR2-related pathway by Mtb. Wild-type Mtb induced an immunosuppressive response of macrophages in an IRAK4- and TRAF6-dependent manner as measured by interleukin 10 production. In conclusion, ChoD is a virulence factor that enables Mtb to disturb the TLR2-related signalling pathway in macrophages and modulate their response.

Regulation of uridine diphosphate-glucuronosyltransferase 1A expression by miRNA-214-5p and miRNA-486-3p

Epigenomics ◽  
2021 ◽  
Author(s):  
Stefan Paulusch ◽  
Sandra Kalthoff ◽  
Steffen Landerer ◽  
Christian Jansen ◽  
Robert Schierwagen ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Potential Risk ◽  
Mrna Levels ◽  
Uridine Diphosphate ◽  
Protein Levels ◽  
Huh7 Cells ◽  
Potential Risk Factors ◽  
Cirrhotic Patients

Aim: This study aimed to identify novel miRNAs (miRs) as regulators of UGT1A gene expression and to evaluate them as potential risk factors for the development of liver fibrosis/cirrhosis. Materials & methods: miRNA target sites in UDP-glucuronosyltransferase 1A (UGT1A) 3′-UTR were predicted and confirmed by luciferase assays, quantitative real-time PCR and western blot using HEK293, HepG2 and Huh7 cells. UGT1A and miRNA expression were analyzed in cirrhotic patients and a mouse model of alcoholic liver fibrosis. Results: miR-214-5p and miR-486-3p overexpression reduced UGT1A mRNA, protein levels and enzyme activity in HepG2 and Huh7 cells. miR-486-3p was upregulated in cirrhotic patients and fibrotic mice livers, whereas UGT1A mRNA levels were reduced. Conclusion: In conclusion, we identified two novel miRNAs capable to repress UGT1A expression in vitro and in vivo. Furthermore, miR-486-3p may represent a potential risk factor for the development or progression of liver fibrosis/cirrhosis by means of a reduced UGT1A-mediated detoxification activity.

Abstract 205: Ouabain Activates the NF-κB Pathway in Macrophages via Na/K-ATPase/TLR4 Complex Leading to Pro-Inflammatory Cytokine Production

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Yiliang Chen ◽  
Roy L Silverstein
Keyword(s):  
Inflammatory Cytokines ◽  
Systemic Inflammation ◽  
Inflammatory Cytokine ◽  
Cytokine Production ◽  
Culture Media ◽  
Mrna Level ◽  
Mrna Levels ◽  
Inflammatory Cytokine Production ◽  
Protein Levels ◽  
Pro Inflammatory Cytokines

Cardiotonic steroids such as ouabain, digoxin, and marinobufagenin are known ligands for the plasma membrane receptor Na/K-ATPase (NKA). These ligands are able to stimulate interaction of the NKA with other membrane and cytosolic proteins leading to cellular events such as activation of kinase cascades and gene transcription. Endogenous cardiotonic steroids have been detected in human circulation and interestingly their levels were elevated in human patients with hypertension, congestive heart failure and diabetes, all of which were associated with chronic systemic inflammation. However, the role of cardiotonic steroids in systemic inflammation and immunity has not been well studied. We previously discovered that ouabain stimulated macrophages to produce pro-inflammatory cytokines, many of which are known targets of the transcription factor, NF-κB. Therefore, we hypothesized that ouabain activates NF-κB pathway leading to pro-inflammatory cytokine production in macrophages. Using Western blot and densitometry analysis, we showed that physiological concentrations of ouabain promoted IκBα degradation (as low as 5 nM ouabain decreased IκBα level by 66.8%±7.4%, n=4). This was accompanied by NF-κB translocation from cytoplasm to the nuclei as shown by immunocytochemistry (% of nuclei NF-κB signals increased from 30.5%±2.3% in control to 62.2%±2.6% in ouabain-treated macrophages, n>25). Moreover, via quantitative RT-PCR (n=3), we found that ouabain increased mRNA levels of pro-inflammatory cytokines such as MCP-1 (3.2±1.1 fold), TNF-α (59.7±35.6 fold), and CXCL-10 (2.8±1.6 fold), all of which are known NF-κB targets. Consistent with the increase in mRNA level, we found that MCP-1 protein levels were elevated in both cell lysates (1.8±0.3 fold) and culture media (1.4±0.1 fold; n=4). Addition of an NF-κB inhibitor blocked MCP-1 production induced by ouabain (n=4). Mechanistically, ouabain stimulated interaction between NKA and TLR4 as shown by Co-Immunoprecipitation (n=3). Blockade of TLR4 signaling using a specific peptide inhibitor, CLI-095, abolished the ouabain effect on NF-κB activation (n=3). We conclude that ouabain activates NF-κB through NKA/TLR4 complex leading to pro-inflammatory cytokine production by macrophages.

scholarly journals Tissue-specific induction of 17 beta-hydroxysteroid dehydrogenase type IV by peroxisome proliferator chemicals is dependent on the peroxisome proliferator-activated receptor alpha

1998 ◽  
Vol 158 (2) ◽  
pp. 237-246 ◽  
Author(s):  
LQ Fan ◽  
RC Cattley ◽  
JC Corton
Keyword(s):  
Hydroxysteroid Dehydrogenase ◽  
Peroxisome Proliferator ◽  
Mrna Levels ◽  
Ppar Alpha ◽  
Peroxisome Proliferator Activated Receptor ◽  
Receptor Alpha ◽  
Protein Levels ◽  
Type Iv ◽  
Liver And Kidney ◽  
The Impact

The 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) family of proteins regulates the levels of the active 17 beta-hydroxy forms of sex steroids. The expression of 17 beta-HSD type IV is induced by peroxisome proliferator chemicals (PPC) in rat liver. In order to characterize more generally the impact of PPC on 17 beta-HSD expression, we determined (1) if expression of other members of the 17 beta-HSD family was coordinately induced by PPC exposure, (2) the tissues in which 17 beta-HSD was induced by PPC, and (3) whether the induction of 17 beta-HSD by PPC was dependent on the peroxisome proliferator-activated receptor alpha (PPAR alpha), the central mediator of PPC effects in the mouse liver. The mRNA levels of 17 beta-HSD I, II, and III were not altered in the liver, kidney, and testis or uterus of rats treated with PPC. The mRNA or 80 kDa a full-length protein levels of 17 beta-HSD IV were strongly induced in liver and kidney, but not induced in adrenals, brown fat, heart, testis, and uterus of rats treated with diverse PPC. In liver and kidneys from treated rats, additional proteins of 66 kDa, 56 kDa, and 32 kDa were also induced which reacted with the anti-17 beta-HSD IV antibodies and were most likely proteolytic fragments of 17 bega-HSD IV. Treatment of mice which lack a functional form of PPAR alpha with PPC, demonstrated that PPC-inducibility of 17 beta-HSD IV mRNA or the 80 kDa protein was dependent on PPAR alpha expression in liver and kidney. Our results demonstrate that 17 beta-HSD IV is induced by PPC through a PPAR alpha-dependent mechanism and support the hypothesis that exposure to PPC leads to alterations in sex steroid metabolism.

scholarly journals AICAR Protects against High Palmitate/High Insulin-Induced Intramyocellular Lipid Accumulation and Insulin Resistance in HL-1 Cardiac Cells by Inducing PPAR-Target Gene Expression

PPAR Research ◽  
2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Ricardo Rodríguez-Calvo ◽  
Manuel Vázquez-Carrera ◽  
Luis Masana ◽  
Dietbert Neumann
Keyword(s):  
Gene Expression ◽  
Insulin Resistance ◽  
Cardiac Cells ◽  
Metabolic Adaptation ◽  
Mrna Levels ◽  
Intramyocellular Lipid ◽  
Protein Levels ◽  
Compound C ◽  
High Insulin ◽  
The Impact

Here we studied the impact of 5-aminoimidazole-4-carboxamide riboside (AICAR), a well-known AMPK activator, on cardiac metabolic adaptation. AMPK activation by AICAR was confirmed by increased phospho-Thr172-AMPK and phospho-Ser79-ACC protein levels in HL-1 cardiomyocytes. Then, cells were exposed to AICAR stimulation for 24 h in the presence or absence of the AMPK inhibitor Compound C, and the mRNA levels of the three PPARs were analyzed by real-time RT-PCR. Treatment with AICAR induced gene expression of all three PPARs, but only thePparaandPpargregulation were dependent on AMPK. Next, we exposed HL-1 cells to high palmitate/high insulin (HP/HI) conditions either in presence or in absence of AICAR, and we evaluated the expression of selected PPAR-targets genes. HP/HI induced insulin resistance and lipid storage was accompanied by increasedCd36,Acot1, andUcp3mRNA levels. AICAR treatment induced the expression ofAcadvlandGlut4, which correlated to prevention of the HP/HI-induced intramyocellular lipid build-up, and attenuation of the HP/HI-induced impairment of glucose uptake. These data support the hypothesis that AICAR contributes to cardiac metabolic adaptationviaregulation of transcriptional mechanisms.

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