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1.
Acta Otolaryngol ; 143(2): 127-133, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36735299

RESUMO

BACKGROUND: Diabetes is associated with inner ear dysfunction. Furthermore, C57BL/6J mice fed high fat diet (HFD), a model for insulin resistance and diabetes, develop endolymphatic hydrops (EH). AIM: Evaluate if betahistine, spironolactone (aldosterone antagonist) and empagliflozin (sodium -glucose cotransporter2 inhibitor) can prevent EH induced by HFD and explore potential mechanisms. METHODS: C57BL/6J mice fed HFD were treated with respective drug. The size of the endolymphatic fluid compartment was measured using contrast enhanced MRI. Secondarily, mice treated with cilostamide, a phosphodiesterase3 inhibitor, to induce EH and HEI-OC1 auditory cells were used to study potential cellular mechanisms of betahistine. RESULTS: HFD-induced EH was prevented by betahistine but not by spironolactone and empagliflozin. Betahistine induced phosphorylation of protein kinaseA substrates but did not prevent cilostamide-induced EH. CONCLUSIONS: Betahistine prevents the development of EH in mice fed HFD, most likely not involving pathways downstream of phosphodiesterase3, an enzyme with implications for dysfunction in diabetes. The finding that spironolactone did not prevent HFD-induced EH suggests different mechanisms for EH induction/treatment since spironolactone prevents EH induced by vasopressin, as previously observed. SIGNIFICANCE: This further demonstrates that independent mechanisms can cause hydropic inner ear diseases which suggests different therapeutic approaches and emphazises the need for personalized medicine.


Assuntos
Diabetes Mellitus , Hidropisia Endolinfática , Resistência à Insulina , Animais , Camundongos , beta-Histina/efeitos adversos , Espironolactona/farmacologia , Espironolactona/uso terapêutico , Camundongos Endogâmicos C57BL , Hidropisia Endolinfática/tratamento farmacológico , Hidropisia Endolinfática/etiologia , Hidropisia Endolinfática/prevenção & controle , Imageamento por Ressonância Magnética
2.
Front Physiol ; 13: 928964, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35991175

RESUMO

AMP-activated protein kinase (AMPK) activation is considered a useful strategy for the treatment of type 2 diabetes (T2D). It is unclear whether the expression and/or activity of AMPK in adipocytes is dysregulated in obesity. Also, the expression/activity pattern of AMPKß isoforms, which are targets for AMPK activators, in adipocytes remains elusive. In this study we show that the two AMPKß isoforms make roughly equal contributions to AMPK activity in primary human and mouse adipocytes, whereas in cultured 3T3-L1 adipocytes of mouse origin and in primary rat adipocytes, ß1-associated activity clearly dominates. Additionally, we found that obesity is not associated with changes in AMPK subunit expression or kinase activity in adipocytes isolated from subcutaneous adipose tissue from individuals with various BMI.

3.
Peptides ; 151: 170747, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35065097

RESUMO

Impaired beta cell function and beta cell death are key features of type 2 diabetes (T2D). Cocaine- and amphetamine-regulated transcript (CART) is necessary for normal islet function in mice. CART increases glucose-stimulated insulin secretion in vivo in mice and in vitro in human islets and CART protects beta cells against glucotoxicity-induced cell death in vitro in rats. Furthermore, beta cell CART is upregulated in T2D patients and in diabetic rodent models as a consequence of hyperglycaemia. The aim of this study was to assess the impact of upregulated beta cell CART on islet hormone secretion and glucose homeostasis in a transgenic mouse model. To this end, mice with beta cell-specific overexpression of CART (CARTtg mice) were generated. CARTtg mice challenged by aging, high fat diet feeding or streptozotocin treatment were phenotyped with respect to in vivo and in vitro insulin and glucagon secretion, glucose homeostasis, and beta cell mass. In addition, the impact of adenoviral overexpression of CART on insulin secretion was studied in INS-1 832/13 cells. CARTtg mice had a normal metabolic phenotype under basal conditions. On the other hand, with age CARTtg mice displayed increased insulin secretion and improved glucose elimination, compared with age-matched WT mice. Furthermore, compared with WT controls, CARTtg mice had increased insulin secretion after feeding a high fat diet, as well as lower glucose levels and higher insulin secretion after streptozotocin treatment. Viral overexpression of CART in INS-1 832/13 cells resulted in increased glucose-stimulated insulin secretion. Together, these results imply that beta cell CART acts to increase insulin secretion when beta cell function is challenged. We propose that the increase in beta cell CART is part of a compensatory mechanisms trying to counteract the hyperglycaemia in T2D.


Assuntos
Diabetes Mellitus Tipo 2 , Hiperglicemia , Resistência à Insulina , Células Secretoras de Insulina , Ilhotas Pancreáticas , Animais , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Glucose/metabolismo , Humanos , Hiperglicemia/genética , Hiperglicemia/metabolismo , Insulina/metabolismo , Resistência à Insulina/genética , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Ratos , Estreptozocina
4.
Acta Otolaryngol ; 142(1): 6-12, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34962430

RESUMO

BACKGROUND: The mechanisms of association between diabetes and inner ear dysfunction are unknown, although endolymphatic hydrops may be involved. We have previously shown that insulin signaling components are expressed in human saccule and that insulin signaling takes place in HEI-OC1 auditory cells. AIM: To explore Nedd4-2 as a target for insulin signaling. MATERIALS AND METHODS: Effects of insulin were analyzed using western blot and confocal microscopy in HEI-OC1 auditory cells. RESULTS: Insulin induced phosphorylation of Nedd4-2 and increased the amount of ENaC at the plasma membrane. Also, protein kinase B (PKB) and NDRG1, a substrate for SGK1 (serum and glucocorticoid stimulated kinase), were phosphorylated in response to insulin. The SGK1 inhibitor GSK650394 prevented insulin-induced phosphorylation of NRDG1, but not of PKB and Nedd4-2, whereas the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin and the PKB inhibitor MK2206 inhibited phosphorylation of all components. Ceramides prevented insulin-induced phosphorylation of PKB and NDRG1, but not of Nedd4-2. The ceramide metabolite sphingosine 1-phosphate induced phosphorylation of Nedd4-2. CONCLUSIONS: Insulin induces phosphorylation of Nedd4-2, most likely involving PI3K/PKB signaling. Sphingosine 1-phosphate might protect Nedd4-2 against ceramide-induced insulin resistance. SIGNIFICANCE: Insulin-mediated regulation of Nedd4-2 might impact on inner ear sodium homeostasis with implications for diabetes-induced inner ear damage.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Canais Epiteliais de Sódio/metabolismo , Insulina/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Repressoras/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Animais , Linhagem Celular , Ceramidas/farmacologia , Orelha Interna/citologia , Fosforilação
5.
Front Physiol ; 12: 740666, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34630160

RESUMO

Lipid uptake can be facilitated via caveolae, specific plasma membrane invaginations abundantly expressed in adipocytes. The dynamin-related protein EH domain-containing 2 (EHD2) stabilizes caveolae at the cell surface. Here, we have examined the importance of EHD2 for lipid handling using primary adipocytes isolated from EHD2 knockout (Ehd2-/- ) C57BL6/N mice. Following high-fat diet (HFD) feeding, we found a clear impairment of epididymal, but not inguinal, adipose tissue expansion in Ehd2-/- compared with Ehd2+/+ (WT) mice. Cell size distribution analysis revealed that Ehd2-/- mice had a lower proportion of small adipocytes, and an accumulation of medium-sized adipocytes in both epididymal and inguinal adipose tissue. Further, PPARγ activity, FABP4 and caveolin-1 expression were decreased in adipocytes isolated from Ehd2-/- mice. Inguinal adipocytes isolated from Ehd2-/- mice displayed reduced lipolysis in response to beta adrenergic receptor agonist, which was associated with reduced phosphorylation of perilipin-1 and hormone sensitive lipase (HSL). This impairment could not be rescued using a cAMP analog, indicating that impaired lipolysis in Ehd2-/- primary adipocytes likely occurs at the level of, or downstream of, protein kinase A (PKA). Altogether, these findings pinpoint the importance of EHD2 for maintained intracellular lipid metabolism, and emphasize differences in mechanisms regulating lipid handling in various adipose-tissue depots.

6.
Biochem J ; 478(3): 633-646, 2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33493298

RESUMO

Activation of AMP-activated protein kinase (AMPK) is considered a valid strategy for the treatment of type 2 diabetes. However, despite the importance of adipose tissue for whole-body energy homeostasis, the effect of AMPK activation in adipocytes has only been studied to a limited extent and mainly with the AMP-mimetic 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR), which has limited specificity. The aim of this study was to evaluate the effect of the allosteric AMPK activators A-769662 and 991 on glucose uptake in adipocytes. For this purpose, primary rat or human adipocytes, and cultured 3T3-L1 adipocytes, were treated with either of the allosteric activators, or AICAR, and basal and insulin-stimulated glucose uptake was assessed. Additionally, the effect of AMPK activators on insulin-stimulated phosphorylation of Akt and Akt substrate of 160 kDa was assessed. Furthermore, primary adipocytes from ADaM site binding drug-resistant AMPKß1 S108A knock-in mice were employed to investigate the specificity of the drugs for the observed effects. Our results show that insulin-stimulated adipocyte glucose uptake was significantly reduced by A-769662 but not 991, yet neither activator had any clear effects on basal or insulin-stimulated Akt/AS160 signaling. The use of AMPKß1 S108A mutant-expressing adipocytes revealed that the observed inhibition of glucose uptake by A-769662 is most likely AMPK-independent, a finding which is supported by the rapid inhibitory effect A-769662 exerts on glucose uptake in 3T3-L1 adipocytes. These data suggest that AMPK activation per se does not inhibit glucose uptake in adipocytes and that the effects of AICAR and A-769662 are AMPK-independent.


Assuntos
Adenilato Quinase/fisiologia , Adipócitos/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Glucose/metabolismo , Pironas/farmacologia , Tiofenos/farmacologia , Células 3T3-L1 , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/metabolismo , Sítio Alostérico , Substituição de Aminoácidos , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Benzimidazóis/farmacologia , Benzoatos/farmacologia , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Feminino , Técnicas de Introdução de Genes , Humanos , Insulina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação de Sentido Incorreto , Fosforilação , Processamento de Proteína Pós-Traducional , Ratos , Ratos Sprague-Dawley , Ribonucleotídeos/farmacologia
7.
Adipocyte ; 9(1): 587-599, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33016185

RESUMO

The aim of this study was to elucidate mechanisms whereby bile acids exert beneficial metabolic effects, using the Cyp8b1-/- mouse as model. These mice are unable to synthesize cholic acid, resulting in increased synthesis of chenodeoxycholic acid and enlarged bile acid pool. Cyp8b1-/- mice were found to be protected against high-fat diet induced obesity. Bomb calorimetry measurements showed increased faecal energy output in Cyp8b1-/ mice. Indirect calorimetry measurements demonstrated increased energy expenditure in Cyp8b1-/- mice. Meal tolerance tests revealed no differences in glucose disposal, but the insulin response was lower in Cyp8b1-/- mice. Intravenous glucose tolerance tests, as well as static incubations of isolated islets, showed no difference between the groups, whereas insulin tolerance tests demonstrated improved insulin sensitivity in Cyp8b1-/- mice. The genes encoding mitochondrial transcription factor A (TFAM) and type 2-iodothyronine deiodinase were upregulated in brown adipose tissue of Cyp8b1/- mice and Western blot analyses showed increased abundance of TFAM, and a trend towards increased abundance of UCP1. The upregulation of TFAM and UCP1 was accompanied by increased mitochondrial density, as shown by transmission electron microscopy. White adipocytes of Cyp8b1-/- mice exhibited increased responsiveness to both catecholamines and insulin in lipolysis experiments and increased insulin-stimulated lipogenesis. In conclusion, increased energy expenditure, mitochondrial density of brown adipocytes and faecal energy output may all contribute to the protection against diet-induced obesity of Cyp8b1-/- mice. Enhanced insulin sensitivity of Cyp8b1-/- mice is accompanied by increased hormonal responsiveness of white adipocytes.


Assuntos
Tecido Adiposo/metabolismo , Dieta Hiperlipídica , Metabolismo Energético , Obesidade/etiologia , Obesidade/metabolismo , Esteroide 12-alfa-Hidroxilase/deficiência , Adipócitos/metabolismo , Tecido Adiposo/patologia , Animais , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Suscetibilidade a Doenças , Glucose/metabolismo , Insulina/metabolismo , Resistência à Insulina , Metabolismo dos Lipídeos , Lipogênese/genética , Lipólise/genética , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Obesidade/patologia
8.
Am J Physiol Endocrinol Metab ; 319(3): E459-E471, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32663099

RESUMO

Insulin resistance in obesity and type 2 diabetes has been shown to be associated with decreased de novo fatty acid (FA) synthesis in adipose tissue. It is known that insulin can acutely stimulate FA synthesis in adipocytes; however, the mechanisms underlying this effect are unclear. The rate-limiting step in FA synthesis is catalyzed by acetyl-CoA carboxylase (ACC), known to be regulated through inhibitory phosphorylation at S79 by the AMP-activated protein kinase (AMPK). Previous results from our laboratory showed an inhibition of AMPK activity by insulin, which was accompanied by PKB-dependent phosphorylation of AMPK at S485. However, whether the S485 phosphorylation is required for insulin-induced inhibition of AMPK or other mechanisms underlie the reduced kinase activity is not known. To investigate this, primary rat adipocytes were transduced with a recombinant adenovirus encoding AMPK-WT or a nonphosphorylatable AMPK S485A mutant. AMPK activity measurements by Western blot analysis and in vitro kinase assay revealed that WT and S485A AMPK were inhibited to a similar degree by insulin, indicating that AMPK S485 phosphorylation is not required for insulin-induced AMPK inhibition. Further analysis suggested an involvement of decreased AMP-to-ATP ratios in the insulin-induced inhibition of AMPK activity, whereas a possible contribution of phosphodiesterases was excluded. Furthermore, we show that insulin-induced AMPK S485 phosphorylation also occurs in human adipocytes, suggesting it to be of an importance yet to be revealed. Altogether, this study increases our understanding of how insulin regulates AMPK activity, and with that, FA synthesis, in adipose tissue.


Assuntos
Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Adipócitos/efeitos dos fármacos , Adipócitos/enzimologia , Insulina/farmacologia , Proteínas Quinases Ativadas por AMP/genética , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Adipócitos/metabolismo , Animais , Metabolismo Energético/efeitos dos fármacos , Ácidos Graxos/metabolismo , Glicerol/metabolismo , Mutação , Diester Fosfórico Hidrolases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley
9.
Artigo em Inglês | MEDLINE | ID: mdl-32238362

RESUMO

OBJECTIVE: The mechanisms underlying the association between diabetes and inner ear dysfunction are not known yet. The aim of the present study is to evaluate the impact of obesity/insulin resistance on inner ear fluid homeostasis in vivo, and to investigate whether the organ of Corti could be a target tissue for insulin signaling using auditory House Ear Institute-Organ of Corti 1 (HEI-OC1) cells as an in vitro model. METHODS: High fat diet (HFD) fed C57BL/6J mice were used as a model to study the impact of insulin resistance on the inner ear. In one study, 12 C57BL/6J mice were fed either control diet or HFD and the size of the inner ear endolymphatic fluid compartment (EFC) was measured after 30 days using MRI and gadolinium contrast as a read-out. In another study, the size of the inner ear EFC was evaluated in eight C57BL/6J mice both before and after HFD feeding, with the same techniques. HEI-OC1 auditory cells were used as a model to investigate insulin signaling in organ of Corti cells. RESULTS: HFD feeding induced an expansion of the EFC in C57BL/6J mice, a hallmark of inner ear dysfunction. Insulin also induced phosphorylation of protein kinase B (PKB/Akt) at Ser473, in a PI3-kinase-dependent manner. The phosphorylation of PKB was inhibited by isoproterenol and IBMX, a general phosphodiesterase (PDE) inhibitor. PDE1B, PDE4D and the insulin-sensitive PDE3B were found expressed and catalytically active in HEI-OC1 cells. Insulin decreased and AICAR, an activator of AMP-activated protein kinase, increased the phosphorylation at the inhibitory Ser79 of acetyl-CoA carboxylase, the rate-limiting enzyme in de novo lipogenesis. Furthermore, the activity of hormone-sensitive lipase, the rate-limiting enzyme in lipolysis, was detected in HEI-OC1 cells. CONCLUSIONS: The organ of Corti could be a target tissue for insulin action, and inner ear insulin resistance might contribute to the association between diabetes and inner ear dysfunction.


Assuntos
Orelha Interna , Resistência à Insulina , Animais , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1 , Insulina , Camundongos , Camundongos Endogâmicos C57BL , Órgão Espiral
10.
Acta Otolaryngol ; 139(8): 685-691, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31145014

RESUMO

Background: The exact pathophysiological mechanism(s) underlying endolymphatic hydrops (EH) remain elusive. We have previously shown that chronic administration of vasopressin and inhibitors of the cAMP/cGMP degrading enzymes (PDE3, PDE4, PDE5) results in the development of EH to mice. Aims/objectives: Evaluate the ability of spironolactone, an aldosterone antagonist, to prevent EH, when induced by different pathways. Material and methods: Mice were treated for 4 weeks with vasopressin, the PDE3 inhibitor cilostamide and the PDE4 inhibitor rolipram in the presence or absence of spironolactone. EH was assessed using high resolution 9.4T MRI. The expression of proteins in human saccule sensory epithelium was studied with immunohistochemistry. Results: Spironolactone prevents EH induced by vasopressin and rolipram, but not hydrops induced by cilostamide. The aldosterone target ENaC and the mineralocorticoid receptor were expressed in the human saccule sensory epithelium. Conclusions: The effect of spironolactone on EH appears to be pathway-dependent and may provide explanations why certain drugs may be effective in some patients with hydropic ear disease while not in others. Significance: Extrapolating this finding to the clinic supports that a personalized medicine approach is probably necessary in the treatment of diseases involving EH, as different pathways may be needed to be targeted for treatment.


Assuntos
Hidropisia Endolinfática/prevenção & controle , Antagonistas de Receptores de Mineralocorticoides/uso terapêutico , Espironolactona/uso terapêutico , Animais , Modelos Animais de Doenças , Hidropisia Endolinfática/induzido quimicamente , Hidropisia Endolinfática/diagnóstico por imagem , Feminino , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos CBA , Quinolonas , Rolipram , Vasopressinas
11.
Cell Signal ; 55: 73-80, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30586628

RESUMO

AIMS/HYPOTHESIS: Salt-inducible kinase 2 (SIK2) is downregulated in adipose tissue from obese or insulin-resistant individuals and inhibition of SIK isoforms results in reduced glucose uptake and insulin signalling in adipocytes. However, the regulation of SIK2 itself in response to insulin in adipocytes has not been studied in detail. The aim of our work was to investigate effects of insulin on various aspects of SIK2 function in adipocytes. METHODS: Primary adipocytes were isolated from human subcutaneous and rat epididymal adipose tissue. Insulin-induced phosphorylation of SIK2 and HDAC4 was analyzed using phosphospecific antibodies and changes in the catalytic activity of SIK2 with in vitro kinase assay. SIK2 protein levels were analyzed in primary adipocytes treated with the proteasome inhibitor MG132. RESULTS: We have identified a novel regulatory pathway of SIK2 in adipocytes, which involves insulin-induced phosphorylation at Thr484. This phosphorylation is impaired in individuals with a reduced insulin action. Insulin stimulation does not affect SIK2 catalytic activity or cellular activity towards HDAC4, but is associated with increased SIK2 protein levels in adipocytes. CONCLUSION/INTERPRETATION: Our data suggest that downregulation of SIK2 in the adipose tissue of insulin-resistant individuals can partially be caused by impaired insulin signalling, which might result in defects in SIK2 expression and function.


Assuntos
Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Resistência à Insulina/fisiologia , Insulina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Adipócitos/citologia , Animais , Células Cultivadas , Humanos , Fosforilação , Ratos , Ratos Sprague-Dawley
12.
Am J Physiol Endocrinol Metab ; 315(5): E1075-E1085, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30253109

RESUMO

Activation of AMP-activated protein kinase (AMPK) is considered an attractive strategy for the treatment of type 2 diabetes. Favorable metabolic effects of AMPK activation are mainly observed in skeletal muscle and liver tissue, whereas the effects in human adipose tissue are only poorly understood. Previous studies, which largely employed the AMPK activator 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR), suggest an antilipolytic role of AMPK in adipocytes. The aim of this work was to reinvestigate the role of AMPK in the regulation of lipolysis, using the novel allosteric small-molecule AMPK activators A-769662 and 991, with a focus on human adipocytes. For this purpose, human primary subcutaneous adipocytes were treated with A-769662, 991, or AICAR, as a control, before being stimulated with isoproterenol. AMPK activity status, glycerol release, and the phosphorylation of hormone-sensitive lipase (HSL), a key regulator of lipolysis, were then monitored. Our results show that both A-769662 and 991 activated AMPK to a level that was similar to, or greater than, that induced by AICAR. In contrast to AICAR, which as expected was antilipolytic, neither A-769662 nor 991 affected lipolysis in human adipocytes, although 991 treatment led to altered HSL phosphorylation. Furthermore, we suggest that HSL Ser660 is an important regulator of lipolytic activity in human adipocytes. These data suggest that the antilipolytic effect observed with AICAR in previous studies is, at least to some extent, AMPK independent.


Assuntos
Adenilato Quinase/metabolismo , Adipócitos/efeitos dos fármacos , Tecido Adiposo/efeitos dos fármacos , Catecolaminas/farmacologia , Lipólise/efeitos dos fármacos , Pironas/farmacologia , Tiofenos/farmacologia , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Compostos de Bifenilo , Feminino , Humanos , Lipólise/fisiologia , Masculino , Camundongos , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Ribonucleotídeos/farmacologia , Esterol Esterase/metabolismo
13.
J Vasc Res ; 54(4): 235-245, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28768281

RESUMO

BACKGROUND: Smooth muscle cells are important for atherosclerotic plaque stability. Their proper ability to communicate with the extracellular matrix is crucial for maintaining the correct tissue integrity. In this study, we have investigated the role of ß-sarcoglycan within the matrix-binding dystrophin-glycoprotein complex in the development of atherosclerosis. RESULTS: Atherosclerotic plaque development was significantly reduced in ApoE-deficient mice lacking ß-sarcoglycan, and their plaques contained an increase in differentiated smooth muscle cells. ApoE-deficient mice lacking ß-sarcoglycan showed a reduction in ovarian adipose tissue and adipocyte size, while the total weight of the animals was not significantly different. Western blot analysis of adipose tissues showed a decreased activation of protein kinase B, while that of AMP-activated kinase was increased in mice lacking ß-sarcoglycan. Analysis of plasma in ß-sarcoglycan-deficient mice revealed reduced levels of leptin, adiponectin, insulin, cholesterol, and triglycerides but increased levels of IL-6, IL-17, and TNF-α. CONCLUSIONS: Our results indicate that the dystrophin-glycoprotein complex and ß-sarcoglycan can affect the atherosclerotic process. Furthermore, the results show the effects of ß-sarcoglycan deficiency on adipose tissue and lipid metabolism, which may also have contributed to the atherosclerotic plaque reduction.


Assuntos
Doenças da Aorta/prevenção & controle , Apolipoproteínas E/deficiência , Aterosclerose/prevenção & controle , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Placa Aterosclerótica , Sarcoglicanas/deficiência , Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/metabolismo , Adipócitos/patologia , Adipocinas/metabolismo , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Aorta Torácica/metabolismo , Aorta Torácica/patologia , Doenças da Aorta/genética , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Complexo de Proteínas Associadas Distrofina/metabolismo , Feminino , Predisposição Genética para Doença , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Fenótipo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sarcoglicanas/genética
14.
Sci Rep ; 7: 40445, 2017 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-28084425

RESUMO

Understanding mechanisms by which a population of beige adipocytes is increased in white adipose tissue (WAT) reflects a potential strategy in the fight against obesity and diabetes. Cyclic adenosine monophosphate (cAMP) is very important in the development of the beige phenotype and activation of its thermogenic program. To study effects of cyclic nucleotides on energy homeostatic mechanisms, mice were generated by targeted inactivation of cyclic nucleotide phosphodiesterase 3b (Pde3b) gene, which encodes PDE3B, an enzyme that catalyzes hydrolysis of cAMP and cGMP and is highly expressed in tissues that regulate energy homeostasis, including adipose tissue, liver, and pancreas. In epididymal white adipose tissue (eWAT) of PDE3B KO mice on a SvJ129 background, cAMP/protein kinase A (PKA) and AMP-activated protein kinase (AMPK) signaling pathways are activated, resulting in "browning" phenotype, with a smaller increases in body weight under high-fat diet, smaller fat deposits, increased ß-oxidation of fatty acids (FAO) and oxygen consumption. Results reported here suggest that PDE3B and/or its downstream signaling partners might be important regulators of energy metabolism in adipose tissue, and potential therapeutic targets for treating obesity, diabetes and their associated metabolic disorders.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Tecido Adiposo Bege/metabolismo , Tecido Adiposo Branco/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/deficiência , Transdução de Sinais , Células 3T3-L1 , Adipócitos/metabolismo , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Metabolismo Energético , Ativação Enzimática , Epididimo/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Obesidade/metabolismo , Obesidade/prevenção & controle , Biogênese de Organelas , Fenótipo , Termogênese , Aumento de Peso
15.
Acta Otolaryngol ; 137(1): 8-15, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27685753

RESUMO

CONCLUSION: The data indicate important roles for phosphodiesterase (PDE) 3, 4, 5, and related cAMP and cGMP pools in the regulation of inner ear fluid homeostasis. Thus, dysfunction of these enzymes might contribute to pathologies of the inner ear. OBJECTIVE: The mechanisms underlying endolymphatic hydrops, a hallmark of inner ear dysfunction, are not known in detail; however, altered balance in cAMP and cGMP signaling systems appears to be involved. Key components of these systems are PDEs, enzymes that modulate the amplitude, duration, termination, and specificity of cAMP and cGMP signaling. METHOD: To evaluate the role of PDE3, 4, and 5 and associated cAMP and cGMP pools in inner ear function, the effect of cilostamide (PDE3 inhibitor), rolipram (PDE4 inhibitor), and sildenafil (PDE5 inhibitor), administrated via mini-osmotic pumps, on mouse inner ear fluid homeostasis was evaluated using 9.4T in vivo MRI in combination with intraperitoneally administered Gadolinium contrast. Also, using human saccule as a model, the expression of PDEs and related signaling molecules and targets was studied using immunohistochemistry. RESULTS: PDE3, PDE4, as well as PDE5 inhibitors resulted in the development of endolymphatic hydrops. Furthermore, PDE3B, PDE4D, and some related signaling components were shown to be expressed in the human saccule.


Assuntos
Hidropisia Endolinfática/enzimologia , Inibidores de Fosfodiesterase , Diester Fosfórico Hidrolases/metabolismo , Sáculo e Utrículo/enzimologia , Animais , Hidropisia Endolinfática/diagnóstico por imagem , Feminino , Humanos , Imageamento por Ressonância Magnética , Camundongos Endogâmicos CBA , Quinolonas , Rolipram , Citrato de Sildenafila
16.
Diabetologia ; 60(2): 314-323, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27807598

RESUMO

AIMS/HYPOTHESIS: Salt-inducible kinases (SIKs) are related to the metabolic regulator AMP-activated protein kinase (AMPK). SIK2 is abundant in adipose tissue. The aims of this study were to investigate the expression of SIKs in relation to human obesity and insulin resistance, and to evaluate whether changes in the expression of SIKs might play a causal role in the development of disturbed glucose uptake in human adipocytes. METHODS: SIK mRNA and protein was determined in human adipose tissue or adipocytes, and correlated to clinical variables. SIK2 and SIK3 expression and phosphorylation were analysed in adipocytes treated with TNF-α. Glucose uptake, GLUT protein levels and localisation, phosphorylation of protein kinase B (PKB/Akt) and the SIK substrate histone deacetylase 4 (HDAC4) were analysed after the SIKs had been silenced using small interfering RNA (siRNA) or inhibited using a pan-SIK-inhibitor (HG-9-91-01). RESULTS: We demonstrate that SIK2 and SIK3 mRNA are downregulated in adipose tissue from obese individuals and that the expression is regulated by weight change. SIK2 is also negatively associated with in vivo insulin resistance (HOMA-IR), independently of BMI and age. Moreover, SIK2 protein levels and specific kinase activity display a negative correlation to BMI in human adipocytes. Furthermore, SIK2 and SIK3 are downregulated by TNF-α in adipocytes. Silencing or inhibiting SIK1-3 in adipocytes results in reduced phosphorylation of HDAC4 and PKB/Akt, less GLUT4 at the plasma membrane, and lower basal and insulin-stimulated glucose uptake in adipocytes. CONCLUSION/INTERPRETATION: This is the first study to describe the expression and function of SIKs in human adipocytes. Our data suggest that SIKs might be protective in the development of obesity-induced insulin resistance, with implications for future treatment strategies.


Assuntos
Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Obesidade/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adulto , Idoso , Animais , Western Blotting , Feminino , Humanos , Resistência à Insulina/genética , Resistência à Insulina/fisiologia , Masculino , Camundongos , Pessoa de Meia-Idade , Fosforilação/efeitos dos fármacos , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator de Necrose Tumoral alfa/farmacologia
17.
Adipocyte ; 5(4): 359-368, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27994949

RESUMO

Short-chain fatty acids (SCFAs), e.g. acetic acid, propionic acid and butyric acid, generated through colonic fermentation of dietary fibers, have been shown to reach the systemic circulation at micromolar concentrations. Moreover, SCFAs have been conferred anti-obesity properties in both animal models and human subjects. Branched SCFAs (BSCFAs), e.g., isobutyric and isovaleric acid, are generated by fermentation of branched amino acids, generated from undigested protein reaching colon. However, BSCFAs have been sparsely investigated when referring to effects on energy metabolism. Here we primarily investigate the effects of isobutyric acid and isovaleric acid on glucose and lipid metabolism in primary rat and human adipocytes. BSCFAs inhibited both cAMP-mediated lipolysis and insulin-stimulated de novo lipogenesis at 10 mM, whereas isobutyric acid potentiated insulin-stimulated glucose uptake by all concentrations (1, 3 and 10 mM) in rat adipocytes. For human adipocytes, only SCFAs inhibited lipolysis at 10 mM. In both in vitro models, BSCFAs and SCFAs reduced phosphorylation of hormone sensitive lipase, a rate limiting enzyme in lipolysis. In addition, BSCFAs and SCFAs, in contrast to insulin, inhibited lipolysis in the presence of wortmannin, a phosphatidylinositide 3-kinase inhibitor and OPC3911, a phosphodiesterase 3 inhibitor in rat adipocytes. Furthermore, BSCFAs and SCFAs reduced insulin-mediated phosphorylation of protein kinase B. To conclude, BSCFAs have effects on adipocyte lipid and glucose metabolism that can contribute to improved insulin sensitivity in individuals with disturbed metabolism.

18.
Cell Signal ; 28(3): 204-213, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26724218

RESUMO

Parathyroid hormone (PTH) is secreted from the parathyroid glands in response to low plasma calcium levels. Besides its classical actions on bone and kidney, PTH may have other important effects, including metabolic effects, as suggested for instance by increased prevalence of insulin resistance and type 2 diabetes in patients with primary hyperparathyroidism. Moreover, secondary hyperparathyroidism may contribute to the metabolic derangements that characterize states of vitamin D deficiency. PTH has been shown to induce adipose tissue lipolysis, but the details of the lipolytic action of PTH have not been described. Here we used primary mouse adipocytes to show that intact PTH (1-84) as well as the N-terminal fragment (1-37) acutely stimulated lipolysis in a dose-dependent manner, whereas the C-terminal fragment (38-84) was without lipolytic effect. The lipolytic action of PTH was paralleled by phosphorylation of known protein kinase A (PKA) substrates, i.e. hormone-sensitive lipase (HSL) and perilipin. The phosphorylation of HSL in response to PTH occurred at the known PKA sites S563 and S660, but not at the non-PKA site S565. PTH-induced lipolysis, as well as phosphorylation of HSL at S563 and S660, was blocked by both the PKA-inhibitor H89 and the adenylate cyclase inhibitor MDL-12330A, whereas inhibitors of extracellular-regulated kinase (ERK), protein kinase B (PKB), AMP-activated protein kinase (AMPK) and Ca(2+)/calmodulin-dependent protein kinase (CaMK) had little or no effect. Inhibition of phosphodiesterase 4 (PDE4) strongly potentiated the lipolytic action of PTH, whereas inhibition of PDE3 had no effect. Our results show that the lipolytic action of PTH is mediated by the PKA signaling pathway with no or minor contribution of other signaling pathways and, furthermore, that the lipolytic action of PTH is limited by simultaneous activation of PDE4. Knowledge of the signaling pathways involved in the lipolytic action of PTH is important for our understanding of how metabolic derangements develop in states of hyperparathyroidism, including vitamin D deficiency.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Lipólise/efeitos dos fármacos , Hormônio Paratireóideo/farmacologia , Esterol Esterase/metabolismo , Inibidores de Adenilil Ciclases/farmacologia , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Proteínas de Transporte/metabolismo , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Humanos , Iminas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Perilipina-1 , Inibidores da Fosfodiesterase 4/farmacologia , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Receptor Tipo 2 de Hormônio Paratireóideo/genética , Receptor Tipo 2 de Hormônio Paratireóideo/metabolismo , Transdução de Sinais/efeitos dos fármacos
19.
Adipocyte ; 4(2): 81-8, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26167409

RESUMO

Fermentation of dietary fibers by colonic microbiota generates short-chain fatty acids (SCFAs), e.g., propionic acid and butyric acid, which have been described to have "anti-obesity properties" by ameliorating fasting glycaemia, body weight and insulin tolerance in animal models. In the present study, we therefore investigate if propionic acid and butyric acid have effects on lipolysis, de novo lipogenesis and glucose uptake in primary rat adipocytes. We show that both propionic acid and butyric acid inhibit isoproterenol- and adenosine deaminase-stimulated lipolysis as well as isoproterenol-stimulated lipolysis in the presence of a phosphodiesterase (PDE3) inhibitor. In addition, we show that propionic acid and butyric acid inhibit basal and insulin-stimulated de novo lipogenesis, which is associated with increased phosphorylation and thus inhibition of acetyl CoA carboxylase, a rate-limiting enzyme in fatty acid synthesis. Furthermore, we show that propionic acid and butyric acid increase insulin-stimulated glucose uptake. To conclude, our study shows that SCFAs have effects on fat storage and mobilization as well as glucose uptake in rat primary adipocytes. Thus, the SCFAs might contribute to healthier adipocytes and subsequently also to improved energy metabolism with for example less circulating free fatty acids, which is beneficial in the context of obesity and type 2 diabetes.

20.
Hear Res ; 330(Pt A): 119-24, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26048336

RESUMO

From histopathological specimens, endolymphatic hydrops has been demonstrated in association with inner ear disorders. Recent studies have observed findings suggestive of hydrops using MRI in humans. Previous studies suggest that vasopressin may play a critical role in endolymph homeostasis and may be involved in the development of Ménière's disease. In this study we evaluate the effect of vasopressin administration in vivo in longitudinal studies using two mouse strains. High resolution MRI at 9.4 T in combination with intraperitoneally delivered Gadolinium contrast, was performed before and after chronic subcutaneous administration of vasopressin via mini-osmotic pumps in the same mouse. A development of endolymphatic hydrops over time could be demonstrated in C57BL6 mice (5 mice, 2 and 4 weeks of administration) as well as in CBA/J mice (4 mice, 2 weeks of administration; 6 mice, 3 and 4 weeks of administration). In most C57BL6 mice hydrops developed first after more than 2 weeks while CBA/J mice had an earlier response. These results may suggest an in vivo model for studying endolymphatic hydrops and corroborates the future use of MRI as a tool in the diagnosis and treatment of inner ear diseases, such as Ménière's disease. MRI may also be developed as a critical tool in evaluating inner ear homeostasis in genetically modified mice, to augment the understanding of human disease.


Assuntos
Orelha Interna/efeitos dos fármacos , Hidropisia Endolinfática/induzido quimicamente , Imageamento por Ressonância Magnética , Vasopressinas/química , Animais , Meios de Contraste/química , Modelos Animais de Doenças , Orelha Interna/fisiopatologia , Edema , Hidropisia Endolinfática/fisiopatologia , Feminino , Gadolínio/química , Homeostase , Infusões Parenterais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Osmose
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