RESUMO
Metabolic changes are critical in the regulation of Ca2+ influx in central and peripheral neuroendocrine cells. To study the regulation of L-type Ca2+ channels by AMPK we used biochemical reagents and ATP/glucose-concentration manipulations in rat chromaffin cells. AICAR and Compound-C, at low concentration, significantly induce changes in L-type Ca2+ channel-current amplitude and voltage dependence. Remarkably, an overlasting decrease in the channel-current density can be induced by lowering the intracellular level of ATP. Accordingly, Ca2+ channel-current density gradually diminishes by decreasing the extracellular glucose concentration. By using immunofluorescence, a decrease in the expression of CaV1.2 is observed while decreasing extracellular glucose, suggesting that AMPK reduces the number of functional Ca2+ channels into the plasma membrane. Together, these results support for the first time the dependence of metabolic changes in the maintenance of Ca2+ channel-current by AMPK. They reveal a key step in Ca2+ influx in secretory cells.
Assuntos
Proteínas Quinases Ativadas por AMP , Aminoimidazol Carboxamida , Canais de Cálcio Tipo L , Células Cromafins , Glucose , Animais , Células Cromafins/metabolismo , Células Cromafins/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Ratos , Glucose/metabolismo , Glucose/farmacologia , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Trifosfato de Adenosina/metabolismo , Ribonucleotídeos/farmacologia , Pirimidinas/farmacologia , Cálcio/metabolismo , Pirazóis/farmacologia , Células Cultivadas , Ratos Wistar , Ativação do Canal Iônico/efeitos dos fármacosRESUMO
Activation of 5' adenosine monophosphate-activated protein kinase (AMPK) stimulates production of the gaseous mediators nitric oxide (NO) and carbon monoxide (CO), which are involved in mucosal defense and gastroprotection. As AMPK itself has gastroprotective effects against several gastric ulcer etiologies, in the present study, we aimed to elucidate whether AMPK may also prevent ethanol-induced injury and play a key role in the associated gastroprotection mediated by hydrogen sulfide (H2S), NO, and CO. Mice were pretreated with AICAR (20â¯mg/kg, an AMPK activator) alone or with 50% ethanol. Other groups were pretreated with respective gaseous mediator inhibitors PAG, l-NAME, or ZnPP IX 30â¯min prior to AICAR, or with gaseous mediator donors NaHS, Lawesson's reagent and l-cysteine (H2S), SNP, l-Arginine (NO), Hemin, or CORM-2 (CO) 30â¯min prior to ethanol with or without compound C (10â¯mg/kg, a non-selective AMPK inhibitor). H2S, nitrate/nitrite (NO3-/NO2-), bilirubin levels, GSH and MDA concentration were evaluated in the gastric mucosa. The gastric mucosa was also collected for histopathological analysis and AMPK expression assessment by immunohistochemistry. Pretreatment with AICAR attenuated the ethanol-induced injury and increased H2S and bilirubin levels but not NO3-/NO2- levels in the gastric mucosa. In addition, inhibition of H2S, NO, or CO synthesis exacerbated the ethanol-induced gastric damage and inhibited the gastroprotection by AICAR. Pretreatment with compound C reversed the gastroprotective effect of NaHS, Lawesson's reagent, l-cysteine, SNP, l-Arginine, CORM-2, or Hemin. Compound C also reversed the effect of NaHS on H2S production, SNP on NO3-/NO2- levels, and Hemin on bilirubin levels. Immunohistochemistry revealed that AMPK is present at basal levels mainly in the gastric mucosa cells, and was increased by pretreatment with NaHS, SNP, and CORM-2. In conclusion, our findings indicate that AMPK activation exerts gastroprotection against ethanol-induced gastric damage and mutually interacts with H2S, NO, or CO to facilitate this process.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Monóxido de Carbono/metabolismo , Gasotransmissores/metabolismo , Sulfeto de Hidrogênio/metabolismo , Óxido Nítrico/metabolismo , Gastropatias/prevenção & controle , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Bilirrubina/metabolismo , Ativação Enzimática , Ativadores de Enzimas/farmacologia , Etanol , Feminino , Mucosa Gástrica/patologia , Masculino , Camundongos , Ribonucleotídeos/farmacologia , Gastropatias/induzido quimicamenteRESUMO
BACKGROUND: Differentiated thyroid carcinomas (DTC) are associated with a good prognosis and a high survival rate. However, tumor recurrence occurs in approximately 20-30% of DTC patients, reinforcing the importance of identifying new molecular targets for cancer management. It has been shown that the 5'-AMP-activated protein kinase (AMPK) is over-activated in papillary thyroid cancer (PTC). This study aimed to investigate the effects of 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR), an AMPK activator, on various aspects of thyroid cancer cell behavior, including cell survival, apoptosis, migration, invasion, and epithelial-to-mesenchymal transition (EMT), in the human thyroid cancer cell lines BCPAP and TPC-1. METHODS: BCPAP and TPC-1 cells were cultivated in Dulbecco's modified Eagle's medium, and the non-tumor-derived cell line Nthy-ORI was grown in RPMI. Cells were treated or not with AICAR for different periods of time. The cell growth rate, cell cycle phase, apoptosis, cell migration, and invasion were analyzed using transwell inserts, and EMT was quantified by the expression of mesenchymal and epithelial markers. RESULTS: AMPK is activated in thyroid cancer cell lines, and AICAR treatment further increased AMPK phosphorylation. After 48 hours of AICAR treatment, the percentage of cells in the G2/M phase decreased, and a G0/G1-phase arrest was induced in both cell lines. AMPK activation effectively induced apoptosis in the BCPAP and TPC-1 cancer cell lines, while no apoptosis induction was observed in Nthy-ORI cells. AICAR also reduced the migration of Nthy-ORI and BCPAP cells by 30% and approximately 60% in TPC-1 cells. AICAR had no effect on cell invasion in Nthy-ORI and TPC-1 cells, but a significant reduction of cell invasion was observed in BCPAP cells. AICAR induced a significant reduction of N-cadherin and no changes in the expression of vimentin or TCF/Zeb1 protein in BCPAP cells. No differences in the expression of EMT markers were found in the AICAR-treated Nthy-ORI cells. A remarkable reduction of vimentin, TCF/Zeb1, and N-cadherin protein expression was detected in the TPC-1 cells. CONCLUSIONS: Increased activation of AMPK in PTC cell lines leads to a strong antitumor response, as measured by the inhibition of cell proliferation, cell migration, and induction of cell death. AMPK activation also reverses EMT in TPC-1 cells.
Assuntos
Proteínas Quinases Ativadas por AMP/efeitos dos fármacos , Aminoimidazol Carboxamida/análogos & derivados , Carcinoma Papilar/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Ribonucleotídeos/farmacologia , Neoplasias da Glândula Tireoide/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Aminoimidazol Carboxamida/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Papilar/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Invasividade Neoplásica , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide/patologiaRESUMO
Chemerin is a novel adipokine associated with obesity and insulin resistance. α-Lipoic acid (α-LA) has shown beneficial properties on diabetes and obesity. The aim of this study was to examine the effects of α-LA on chemerin production in adipocytes in absence or presence of TNF-α, insulin and AICAR. The potential signaling pathways involved in α-LA effects on chemerin were also analyzed. α-LA actions on chemerin were tested in differentiated 3T3-L1 adipocytes and in some cases in human subcutaneous and omental adipocytes. Chemerin mRNA levels were measured by RT-PCR and the amount of chemerin secreted to culture media was determined by ELISA. α-LA induced a concentration-dependent inhibition on both chemerin secretion and mRNA levels in 3T3-L1 adipocytes. The AMPK activator AICAR and the PI3K inhibitor LY294002 dramatically abrogated both chemerin secretion and gene expression, and further potentiated the inhibitory effect of α-LA on chemerin secretion. Insulin was able to partially reverse the inhibitory action of α-LA on chemerin secretion. α-LA also reduced basal chemerin secretion in both subcutaneous and omental adipocytes from overweight/obese subjects. Moreover, α-LA was able to abolish the stimulatory effects of the pro-inflammatory cytokine TNF-α on chemerin secretion. Our data demonstrated the ability of α-LA to inhibit chemerin production, an adipokine associated to obesity and metabolic syndrome, suggesting that the reduction of chemerin could contribute to the antiobesity/antidiabetic properties described for α-LA.
Assuntos
Adipócitos/efeitos dos fármacos , Fármacos Antiobesidade/farmacologia , Quimiocinas/metabolismo , Hipoglicemiantes/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ácido Tióctico/farmacologia , Células 3T3-L1 , Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Quimiocinas/sangue , Quimiocinas/genética , Relação Dose-Resposta a Droga , Regulação para Baixo , Humanos , Insulina/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Camundongos , PPAR gama/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Ratos Wistar , Ribonucleotídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transfecção , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Cellular energy regulation relies on complex signaling pathways that respond to fuel availability and metabolic demands. Dysregulation of these networks is implicated in the development of human metabolic diseases such as obesity and metabolic syndrome. In Caenorhabditis elegans the AMP-activated protein kinase, AAK, has been associated with longevity and stress resistance; nevertheless its precise role in energy metabolism remains elusive. In the present study, we find an evolutionary conserved role of AAK in oxidative metabolism. Similar to mammals, AAK is activated by AICAR and metformin and leads to increased glycolytic and oxidative metabolic fluxes evidenced by an increase in lactate levels and mitochondrial oxygen consumption and a decrease in total fatty acids and lipid storage, whereas augmented glucose availability has the opposite effects. We found that these changes were largely dependent on the catalytic subunit AAK-2, since the aak-2 null strain lost the observed metabolic actions. Further results demonstrate that the effects due to AAK activation are associated to SBP-1 and NHR-49 transcriptional factors and MDT-15 transcriptional co-activator, suggesting a regulatory pathway that controls oxidative metabolism. Our findings establish C. elegans as a tractable model system to dissect the relationship between distinct molecules that play a critical role in the regulation of energy metabolism in human metabolic diseases.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas Serina-Treonina Quinases/genética , Subunidades Proteicas/genética , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição/genética , Proteínas Quinases Ativadas por AMP , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Respiração Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Humanos , Ácido Láctico/metabolismo , Longevidade/genética , Metformina/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oxigênio/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Subunidades Proteicas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Ribonucleotídeos/farmacologia , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2±2 vs 7.9±1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4±2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3±2 vs 7.5±2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1±2 vs 7.4±2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca2+/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.
Assuntos
Animais , Masculino , Músculo Liso Vascular/fisiologia , Cadeias Leves de Miosina/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Vasoconstrição/fisiologia , Aorta Torácica , Acetilglucosamina/análogos & derivados , Acetilglucosamina/farmacologia , Acilação/efeitos dos fármacos , Acilação/fisiologia , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Azepinas/farmacologia , Western Blotting , Inibidores Enzimáticos/farmacologia , Hipoglicemiantes/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Oxazóis/farmacologia , Oximas/farmacologia , Fenilcarbamatos/farmacologia , Fenilefrina/agonistas , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Ratos Wistar , Ribonucleotídeos/farmacologia , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , beta-N-Acetil-Hexosaminidases/antagonistas & inibidoresRESUMO
O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2 ± 2 vs 7.9 ± 1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4 ± 2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3 ± 2 vs 7.5 ± 2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1 ± 2 vs 7.4 ± 2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca(2+)/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.
Assuntos
Músculo Liso Vascular/fisiologia , Cadeias Leves de Miosina/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Vasoconstrição/fisiologia , Acetilglucosamina/análogos & derivados , Acetilglucosamina/farmacologia , Acilação/efeitos dos fármacos , Acilação/fisiologia , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Aorta Torácica , Azepinas/farmacologia , Western Blotting , Inibidores Enzimáticos/farmacologia , Hipoglicemiantes/farmacologia , Masculino , Toxinas Marinhas , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Oxazóis/farmacologia , Oximas/farmacologia , Fenilcarbamatos/farmacologia , Fenilefrina/agonistas , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Ratos Wistar , Ribonucleotídeos/farmacologia , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , beta-N-Acetil-Hexosaminidases/antagonistas & inibidoresRESUMO
BACKGROUND: Glucose is transported into cells by specific glucose transporter proteins (GLUTs) that are widely expressed in a tissue-specific manner. The mechanisms that regulate glucose uptake and metabolism in thyroid cells are poorly defined. Recently, our group showed that AMP-activated protein kinase (AMPK) plays a pivotal role in the rat thyroid gland, downregulating iodide uptake by thyroid cells even in the presence of its main stimulator thyrotropin (TSH). Since AMPK increases glucose uptake in different tissues, and taken into consideration that in pathophysiological conditions such as thyroid cancer a negative correlation between iodide and glucose uptake occurs, we hypothesized that AMPK might modulate glucose uptake in thyroid cells. METHODS: Rat follicular thyroid PCCL3 cells cultivated in Ham's F-12 supplemented with 5% calf serum and hormones were exposed to the AMPK pharmacological activator 5-aminoimidazole-4 carboxamide ribonucleoside (AICAR) or AMPK antagonist compound C for 24 hours either in the presence or absence of TSH. Glucose uptake was assessed in vitro using 2-deoxy-D-[(3)H]glucose. RESULTS: AMPK activation by AICAR induced a significant increase in glucose uptake by PCCL3 cells, an effect that was completely reversed by the AMPK inhibitor compound C. Also, the AICAR mediated increase in glucose uptake was detected either in the presence or absence of TSH. The mechanism by which AICAR increases glucose uptake is related to higher levels of GLUT 1 protein content and hexokinase (HK) activity in thyroid cells. CONCLUSION: Our results show that AMPK activation significantly upregulates GLUT 1 content and glucose uptake, and it also stimulates hexokinase activity, the first step of glycolysis.
Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Glucose/metabolismo , Glândula Tireoide/metabolismo , Tireotropina/fisiologia , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Células Cultivadas , Transportador de Glucose Tipo 1/biossíntese , Pirazóis/farmacologia , Pirimidinas/farmacologia , Ratos , Ribonucleotídeos/farmacologia , Glândula Tireoide/efeitos dos fármacos , Regulação para CimaRESUMO
AtFBS1 is an F-box protein whose transcript accumulates in response to biotic and abiotic stresses. Previous evidence suggests that a postranscriptional event regulates AtFBS1 expression [1]. We now found that AtFBS1 interacts with 14-3-3 proteins through its amino-terminus and the F-box motif. Deletion of any of these regions abolishes the interaction between AtFBS1 and 14-3-3 proteins. On the other hand, the treatment with the proteasome inhibitor MG132 or the deletion of the F-box from AtFBS1 increases ß-glucuronidase (GUS) activity in plants containing a translational fusion of AtFBS1 with the GUS reporter gene, indicating that AtFBS1 is degraded by the 26S proteasome. MG132 treatment of seedlings containing a gene fusion between AtFBS1 and the TAP (Tandem Affinity Purification) cassette causes an increase in the half-life of the protein. In an attempt to understand the role of 14-3-3 interactions, we treated Arabidopsis seedlings with 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranosyl 5'-monophosphate (AICAR), an inhibitor of 14-3-3 client interactions. We observed an increase in AtFBS1-TAP stability as a consequence of AICAR treatment. Based on these data we propose that 14-3-3 proteins promote the dimerization of SCF(AtFBS1). This also may enhance the AtFBS1 autoubiquitination activity and its degradation by the 26S proteasome. AICAR also affects Cullin1 (CUL1) modification by RUB1, which would provide an additional element to the effect of this compound on AtFBS1 stability.
Assuntos
Proteínas 14-3-3/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas F-Box/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ribonucleotídeos/farmacologia , Estresse Fisiológico , Proteínas 14-3-3/antagonistas & inibidores , Adaptação Fisiológica , Aminoimidazol Carboxamida/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas Culina/metabolismo , Motivos F-Box , Genes de Plantas , Glucuronidase/genética , Glucuronidase/metabolismo , Leupeptinas/farmacologia , Inibidores de Proteassoma/farmacologia , Multimerização Proteica , Estabilidade Proteica , Proteólise , Plântula/efeitos dos fármacos , Plântula/metabolismo , Ubiquitinação , Ubiquitinas/metabolismoRESUMO
The aim of this study was to investigate the role of AMP-kinase (AMPK) in the regulation of iodide uptake by the thyroid gland. Iodide uptake was assessed in PCCL3 follicular thyroid cells exposed to the AMPK agonist 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR), and also in rat thyroid glands 24 h after a single intraperitoneal injection of AICAR. In PCCL3 cells, AICAR-induced AMPK and acetyl-CoA carboxylase (ACC) phosphorylation decreased iodide uptake in a concentration-dependent manner, while the AMPK inhibitor compound C prevented this effect. In the thyroid gland of rats injected with AICAR, AMPK and ACC phosphorylation was increased and iodide uptake was reduced by ~35%. Under conditions of increased AMPK phosphorylation/activation such as TSH deprivation or AICAR treatment, significant reductions in cellular Na(+)/I(-)-symporter (NIS) protein (~41%) and mRNA content (~65%) were observed. The transcriptional (actinomycin D) and translational (cycloheximide) inhibitors, as well as the AMPK inhibitor compound C prevented AICAR-induced reduction of NIS protein content in PCCL3 cells. The presence of TSH in the culture medium reduced AMPK phosphorylation in PCCL3 cells, while inhibition of protein kinase A (PKA) with H89 prevented this effect. Conversely, the adenylyl cyclase activator forskolin abolished the AMPK phosphorylation response induced by TSH withdrawal in PCCL3 cells. These findings demonstrate that TSH suppresses AMPK phosphorylation/activation in a cAMP-PKA-dependent manner. In summary, we provide novel evidence that AMPK is involved in the physiological regulation of iodide uptake, which is an essential step for the formation of thyroid hormones as well as for the regulation of thyroid function.