RESUMO
AIMS/HYPOTHESIS: Glucagon-stimulated hepatic gluconeogenesis contributes to endogenous glucose production during fasting. Recent studies suggest that TGF-ß is able to promote hepatic gluconeogenesis in mice. However, the physiological relevance of serum TGF-ß levels to human glucose metabolism and the mechanism by which TGF-ß enhances gluconeogenesis remain largely unknown. As enhanced gluconeogenesis is a signature feature of type 2 diabetes, elucidating the molecular mechanisms underlying TGF-ß-promoted hepatic gluconeogenesis would allow us to better understand the process of normal glucose production and the pathophysiology of this process in type 2 diabetes. This study aimed to investigate the contribution of upregulated TGF-ß1 in human type 2 diabetes and the molecular mechanism underlying the action of TGF-ß1 in glucose metabolism. METHODS: Serum levels of TGF-ß1 were measured by ELISA in 74 control participants with normal glucose tolerance and 75 participants with type 2 diabetes. Human liver tissue was collected from participants without obesity and with or without type 2 diabetes for the measurement of TGF-ß1 and glucagon signalling. To investigate the role of Smad3, a key signalling molecule downstream of the TGF-ß1 receptor, in mediating the effect of TGF-ß1 on glucagon signalling, we generated Smad3 knockout mice. Glucose levels in Smad3 knockout mice were measured during prolonged fasting and a glucagon tolerance test. Mouse primary hepatocytes were isolated from Smad3 knockout and wild-type (WT) mice to investigate the underlying molecular mechanisms. Smad3 phosphorylation was detected by western blotting, levels of cAMP were detected by ELISA and levels of protein kinase A (PKA)/cAMP response element-binding protein (CREB) phosphorylation were detected by western blotting. The dissociation of PKA subunits was measured by immunoprecipitation. RESULTS: We observed higher levels of serum TGF-ß1 in participants without obesity and with type 2 diabetes than in healthy control participants, which was positively correlated with HbA1c and fasting blood glucose levels. In addition, hyperactivation of the CREB and Smad3 signalling pathways was observed in the liver of participants with type 2 diabetes. Treating WT mouse primary hepatocytes with TGF-ß1 greatly potentiated glucagon-stimulated PKA/CREB phosphorylation and hepatic gluconeogenesis. Mechanistically, TGF-ß1 treatment induced the binding of Smad3 to the regulatory subunit of PKA (PKA-R), which prevented the association of PKA-R with the catalytic subunit of PKA (PKA-C) and led to the potentiation of glucagon-stimulated PKA signalling and gluconeogenesis. CONCLUSIONS/INTERPRETATION: The hepatic TGF-ß1/Smad3 pathway sensitises the effect of glucagon/PKA signalling on gluconeogenesis and synergistically promotes hepatic glucose production. Reducing serum levels of TGF-ß1 and/or preventing hyperactivation of TGF-ß1 signalling could be a novel approach for alleviating hyperglycaemia in type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2 , Hiperglicemia , Humanos , Animais , Camundongos , Glucagon/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Hiperglicemia/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Hepatócitos/metabolismo , Fígado/metabolismo , Glucose/metabolismo , Gluconeogênese , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Camundongos Knockout , Camundongos Endogâmicos C57BLRESUMO
After confirmation of the presence of adiponectin (ADPN) receptors and intra-cellular binding proteins in coronary artery smooth muscle cells (VSMC), we tested the hypotheses that, in acute insulin resistance: (i) the activation/inactivation of metabolic and mitogenic insulin signaling pathways are inversely affected by ADPN and, (ii) changes in VSMC migration/proliferation rates correlate with signal activity/inactivity. In primary cultures of VSMC exposed to high glucose and palmitate plus insulin, the expression of PI-3 kinase (Akt and m-TOR), MAP-Kinase (Erk and p-38) molecules, and inflammatory markers (TLR-4 and IkB-α) were assessed with Western blot, in the absence/presence of AdipoRon (AR). Migration and proliferation rates were measured in similar experimental conditions. There were decreases of ~ 25% (p-Akt) and 40-60% (p-mTOR) expressions with high glucose/palmitate, which reversed when AR was added were. Elevations in p-Erk and p-p38 expressions were obliterated by AR. Although, no changes were detected with high glucose and palmitate, when AR was added, a decline in inflammatory activity was substantiated by a ~ 50% decrease in TLR-4 and 40-60% increase in IkBα expression. Functional assays showed 10-20% rise in VSMC proliferation with high glucose and palmitate, but addition of AR lead to 15-25% decline. The degree of VSMC migration was reduced with AR addition by ~ 15%, ~ 35% and 55%, in VSMC exposed to 5 mM, 25 mM glucose and 25 mM + 200 µM palmitate, respectively. Changes in intracellular molecular messaging in experiments mimicking acute insulin resistance suggest that anti-inflammatory and anti-atherogenic actions of ADPN in VSMC are mediated via insulin signaling pathways.
Assuntos
Adiponectina/metabolismo , Insulina/isolamento & purificação , Insulina/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Piperidinas/farmacologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Glucose/farmacologia , Humanos , Proteínas I-kappa B/metabolismo , Inflamação/metabolismo , Insulina/metabolismo , Palmitatos/farmacologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Adiponectina/agonistas , Serina-Treonina Quinases TOR/metabolismo , Receptor 4 Toll-Like/metabolismo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Chronic inflammation in adipose tissue plays a key role in obesity-induced insulin resistance. However, the mechanisms underlying obesity-induced inflammation remain elusive. Here we show that obesity promotes mtDNA release into the cytosol, where it triggers inflammatory responses by activating the DNA-sensing cGAS-cGAMP-STING pathway. Fat-specific knockout of disulfide-bond A oxidoreductase-like protein (DsbA-L), a chaperone-like protein originally identified in the mitochondrial matrix, impaired mitochondrial function and promoted mtDNA release, leading to activation of the cGAS-cGAMP-STING pathway and inflammatory responses. Conversely, fat-specific overexpression of DsbA-L protected mice against high-fat diet-induced activation of the cGAS-cGAMP-STING pathway and inflammation. Taken together, we identify DsbA-L as a key molecule that maintains mitochondrial integrity. DsbA-L deficiency promotes inflammation and insulin resistance by activating the cGAS-cGAMP-STING pathway. Our study also reveals that, in addition to its well-characterized roles in innate immune surveillance, the cGAS-cGAMP-STING pathway plays an important role in mediating obesity-induced metabolic dysfunction.
Assuntos
DNA Mitocondrial/metabolismo , Glutationa Transferase/genética , Resistência à Insulina , Proteínas de Membrana/genética , Nucleotidiltransferases/genética , Obesidade/genética , Células 3T3-L1 , Adipócitos/metabolismo , Adipócitos/patologia , Animais , Dieta Hiperlipídica/efeitos adversos , Regulação da Expressão Gênica , Glutationa Transferase/deficiência , Humanos , Inflamação , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Nucleotidiltransferases/metabolismo , Obesidade/etiologia , Obesidade/metabolismo , Obesidade/patologia , Cultura Primária de Células , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transdução de SinaisRESUMO
Adiponectin is an adipokine that can exert a regulatory function on bone metabolism. However, there are many contradictions between clinical and pre-clinical studies on adiponectin. APPL1 is an adaptor protein that can interact with adiponectin receptors. In the current study, we found that knockout of the Appl1 gene in male mice was associated with higher bone volume and numbers of trabeculae than in females or controls. The trabecular thickness, cortical thickness, ratio of bone volume/trabecular volume, cross-sectional bone area, and mean polar moment of inertia increased in Appl1 KO mice compared with wild-type mice. The number of osteoblasts increased but the number of adipocytes decreased in Appl1 KO mice. Knockdown of Appl1 impaired adipogenesis in bone marrow-derived mesenchymal stem cells. Mineralization was increased by knockdown of Appl1 during osteoblast differentiation. Data from differentiation-related genes showed results consistent with the in vivo effects. In summary, this study provides further clarification of the effect of the adiponectin signaling pathway on bone metabolism.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células-Tronco Mesenquimais/citologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Adipócitos/citologia , Adipogenia/genética , Animais , Diferenciação Celular/genética , Técnicas de Inativação de Genes , Masculino , Camundongos , Osteoblastos/citologiaRESUMO
Adiponectin is an adipocyte-derived hormone with antidiabetic activities that include increasing the sensitivity of cells to insulin. Adaptor protein containing pleckstrin homology domain, phosphotyrosine-binding domain, and leucine zipper motif (APPL1) stimulates adiponectin signaling and promotes adiponectin's insulin-sensitizing effects by binding to two adiponectin receptors, AdipoR1 and AdipoR2, and the insulin receptor. In this study, we report an alternative splicing variant of APPL1 (APPL1sv) that is highly expressed in mouse liver, pancreas, and spleen tissues. The expression levels of APPL1sv in liver tissues were enhanced in a mouse model of obesity and diabetic dyslipidemia (i.e. db/db mice) and reduced in calorie-restricted mice compared with ad libitum-fed mice. APPL1sv overexpression or suppression inhibited or enhanced, respectively, adiponectin-stimulated phosphorylation of AMP protein kinase (AMPK) in mouse hepatocytes. We also found that APPL1sv binds to AdipoR1 and AdipoR2 under basal conditions and that adiponectin treatment reduces this binding. Overexpression of APPL1sv blocked adiponectin-induced interactions of APPL1 with the adiponectin receptors. Moreover, adenovirus-mediated and short hairpin RNA-based suppression of APPL1sv greatly reduced high fat diet-induced insulin resistance and hepatic glucose production in mice. Our study identifies a key suppressor of hepatic adiponectin signaling and insulin sensitivity, a finding that may shed light on identifying effective therapeutic targets for treating insulin resistance and type 2 diabetes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Adiponectina/metabolismo , Processamento Alternativo , Resistência à Insulina , Fígado/metabolismo , Obesidade/genética , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células Cultivadas , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Dislipidemias/genética , Dislipidemias/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Regulação para CimaRESUMO
Ectopic fat deposition (EFD) in the kidney has been shown to play a causal role in diabetic nephropathy; however, the mechanism underlying EFD remains elusive. By transcriptome analysis, we found decreased expression levels of disulfide-bond A oxidoreductase-like protein (DsbA-L) in the kidneys of diabetic mice (induced by high-fat diet plus Streptozotocin) compared with control mice. Increased expression of adipocyte differentiation-related protein and abnormal levels of collagen I, fibronectin, and phosphorylated 5'AMP-activated kinase (p-AMPK), adipose triglyceride lipase (p-ATGL), and HMG-CoA reductase (p-HMGCR) were also observed in diabetic mice. These alterations were accompanied by deposition of lipid droplets in the kidney, and were more pronounced in diabetic DsbA-L knockout mice. In vitro, overexpression of DsbA-L ameliorated high glucose-induced intracellular lipid droplet deposition in a human proximal tubular cell line, and DsbA-L siRNA aggravated lipid droplet deposition and reduced the levels of p-AMPK, p-ATGL, and p-HMGCR. High glucose and palmitic acid treatment enhanced the expression of interleukin-1ß and interleukin-18; these enhancements were further increased after treatment with DsbA-L siRNA but alleviated by co-treatment with an AMPK activator. In kidney biopsy tissue from patients with diabetic nephropathy, DsbA-L expression was negatively correlated with EFD and tubular damage. Collectively, these results suggest that DsbA-L has a protective role against EFD and lipid-related kidney damage in diabetic nephropathy. Activation of the AMPK pathway is a potential mechanism underlying DsbA-L action in the kidney.
Assuntos
Nefropatias Diabéticas/patologia , Glutationa Transferase/metabolismo , Rim/patologia , Metabolismo dos Lipídeos , Adenilato Quinase/metabolismo , Adulto , Animais , Biópsia , Linhagem Celular , Colesterol/biossíntese , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/metabolismo , Dieta Hiperlipídica/efeitos adversos , Feminino , Glutationa Transferase/genética , Humanos , Hidroximetilglutaril-CoA Redutases/metabolismo , Rim/citologia , Gotículas Lipídicas/patologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , RNA Interferente Pequeno/metabolismo , Estreptozocina/toxicidadeRESUMO
Hepatic insulin resistance and hepatosteatosis in diet-induced obesity are associated with various metabolic diseases, yet the underlying mechanisms remain to be fully elucidated. Here we show that the expression levels of the disulfide-bond A oxidoreductase-like protein (DsbA-L) are significantly reduced in the liver of obese mice and humans. Liver-specific knockout or adenovirus-mediated overexpression of DsbA-L exacerbates or alleviates, respectively, high-fat diet-induced mitochondrial dysfunction, hepatosteatosis, and insulin resistance in mice. Mechanistically, we found that DsbA-L is localized in mitochondria and that its deficiency is associated with impairment of maximum respiratory capacity, elevated cellular oxidative stress, and increased JNK activity. Our results identify DsbA-L as a critical regulator of mitochondrial function, and its down-regulation in the liver may contribute to obesity-induced hepatosteatosis and whole body insulin resistance.-Chen, H., Bai, J., Dong, F., Fang, H., Zhang, Y., Meng, W., Liu, B., Luo, Y., Liu, M., Bai, Y., Abdul-Ghani, M. A., Li, R., Wu, J., Zeng, R., Zhou, Z., Dong, L. Q., Liu, F. Hepatic DsbA-L protects mice from diet-induced hepatosteatosis and insulin resistance.
Assuntos
Gorduras na Dieta/efeitos adversos , Fígado Gorduroso/induzido quimicamente , Glutationa Transferase/metabolismo , Resistência à Insulina/fisiologia , Fígado/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Dieta Hiperlipídica/efeitos adversos , Regulação Enzimológica da Expressão Gênica , Técnica Clamp de Glucose , Glutationa Transferase/genética , Hepatócitos , Humanos , Fígado/enzimologia , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias Hepáticas/metabolismo , Estresse Oxidativo , Consumo de OxigênioRESUMO
Adiponectin is an adipokine with insulin-sensitizing and anti-inflammatory functions. We previously reported that adiponectin multimerization and stability are promoted by the disulfide bond A oxidoreductase-like protein (DsbA-L) in cells and in vivo. However, the precise mechanism by which DsbA-L regulates adiponectin biosynthesis remains elusive. Here we show that DsbA-L is co-localized with the endoplasmic reticulum (ER) marker protein disulfide isomerase and the mitochondrial marker MitoTracker. In addition, DsbA-L interacts with the ER chaperone protein Ero1-Lα in 3T3-L1 adipocytes. In silico analysis and truncation mapping studies revealed that DsbA-L contains an ER targeting signal at its N terminus. Deletion of the first 6 residues at the N terminus greatly impaired DsbA-L localization in the ER. Overexpression of the wild type but not the ER localization-defective mutant of DsbA-L protects against thapsigargin-induced ER stress and adiponectin down-regulation in 3T3-L1 adipocytes. In addition, overexpression of the wild type but not the ER localization-defective mutant of DsbA-L promotes adiponectin multimerization. Together, our results reveal that DsbA-L is localized in both the mitochondria and the ER in adipocytes and that its ER localization plays a critical role in suppressing ER stress and promoting adiponectin biosynthesis and secretion.
Assuntos
Adipócitos/metabolismo , Adiponectina/genética , Estresse do Retículo Endoplasmático/genética , Glutationa Transferase/genética , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adiponectina/metabolismo , Animais , Diferenciação Celular , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação da Expressão Gênica , Glutationa Transferase/metabolismo , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mutação , Oxirredutases/genética , Oxirredutases/metabolismo , Multimerização Proteica , Transdução de Sinais , Tapsigargina/farmacologiaRESUMO
Although Appl1 and Appl2 have been implicated in multiple cellular activities, we and others have found that Appl1 is dispensable for mouse embryonic development, suggesting that Appl2 can substitute for Appl1 during development. To address this possibility, we generated conditionally targeted Appl2 mice. We found that ubiquitous Appl2 knockout (Appl2-/-) mice, much like Appl1-/- mice, are viable and grow normally to adulthood. Intriguingly, when Appl1-/- mice were crossed with Appl2-/- mice, we found that homozygous Appl1;Appl2 double knockout (DKO) animals are also viable and grossly normal with regard to reproductive potential and postnatal growth. Appl2-null and DKO mice were found to exhibit altered red blood cell physiology, with erythrocytes from these mice generally being larger and having a more irregular shape than erythrocytes from wild type mice. Although Appl1/2 proteins have been previously shown to have a very strong interaction with phosphatidylinositol-3 kinase (Pi3k) in thymic T cells, Pi3k-Akt signaling and cellular differentiation was unaltered in thymocytes from Appl1;Appl2 (DKO) mice. However, Appl1/2-null mouse embryonic fibroblasts exhibited defects in HGF-induced Akt activation, migration, and invasion. Taken together, these data suggest that Appl1 and Appl2 are required for robust HGF cell signaling but are dispensable for embryonic development and reproduction.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Alelos , Animais , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Embrião de Mamíferos/citologia , Ativação Enzimática/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Marcação de Genes , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Camundongos , Camundongos Knockout , Células-Tronco Embrionárias Murinas/citologia , Organogênese/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Reprodução , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
Adiponectin (APN) is an adipocyte-secreted adipokine that exerts well-characterized antidiabetic properties. Patients with type 2 diabetes (T2D) are characterized by reduced APN levels in circulation and impaired stem cell and progenitor cell mobilization from the bone marrow for tissue repair and remodeling. In this study, we found that APN regulates the mobilization and recruitment of bone marrow-derived mesenchymal stem cells (BMSCs) to participate in tissue repair and regeneration. APN facilitated BMSCs migrating from the bone marrow into the circulation to regenerate bone by regulating stromal cell-derived factor (SDF)-1 in a mouse bone defect model. More importantly, we found that systemic APN infusion ameliorated diabetic mobilopathy of BMSCs, lowered glucose concentration, and promoted bone regeneration in diet-induced obesity mice. In vitro studies allowed us to identify Smad1/5/8 as a novel signaling mediator of APN receptor (AdipoR)-1 in BMSCs and osteoblasts. APN stimulation of MC3T3-E1 osteoblastic cells led to Smad1/5/8 phosphorylation and nuclear localization and increased SDF-1 mRNA expression. Although APN-mediated phosphorylation of Smad1/5/8 occurred independently from adaptor protein, phosphotyrosine interaction, pleckstrin homology domain, and leucine zipper containing 1, it correlated with the disassembly of protein kinase casein kinase 2 and AdipoR1 in immunoprecipitation experiments. Taken together, this study identified APN as a regulator of BMSCs migration in response to bone injury. Therefore, our findings suggest APN signaling could be a potential therapeutic target to improve bone regeneration and homeostasis, especially in obese and T2D patients.
Assuntos
Adiponectina/metabolismo , Doenças Ósseas/terapia , Células da Medula Óssea/citologia , Diabetes Mellitus Tipo 2/terapia , Células-Tronco Mesenquimais/citologia , Nicho de Células-Tronco/fisiologia , Células 3T3 , Animais , Doenças Ósseas/metabolismo , Doenças Ósseas/patologia , Células da Medula Óssea/metabolismo , Proliferação de Células/fisiologia , Quimiocina CXCL12/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores CXCR4/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
Adiponectin (APN), the most abundant adipocyte-secreted adipokine, regulates energy homeostasis and exerts well-characterized insulin-sensitizing properties. The peripheral or central effects of APN regulating bone metabolism are beginning to be explored but are still not clearly understood. In the present study, we found that APN-knockout (APN-KO) mice fed a normal diet exhibited decreased trabecular structure and mineralization and increased bone marrow adiposity compared with wild-type (WT) mice. APN intracerebroventricular infusions decreased uncoupling protein 1 (UCP1) expression in brown adipose tissue, epinephrine and norepinephrine serum levels, and osteoclast numbers, whereas osteoblast osteogenic marker expression and trabecular bone mass increased in APN-KO and WT mice. In addition, centrally administered APN increased hypothalamic tryptophan hydroxylase 2 (TPH2), cocaine- and amphetamine-regulated transcript (CART), and 5-hydroxytryptamine (serotonin) receptor 2C (Htr2C) expressions but decreased hypothalamic cannabinoid receptor-1 expression. Treatment of immortalized mouse neurons with APN demonstrated that APN-mediated effects on TPH2, CART, and Htr2C expression levels were abolished by downregulating adaptor protein containing pleckstrin homology domain, phosphotyrosine domain, and leucine zipper motif (APPL)-1 expression. Pharmacological increase in sympathetic activity stimulated adipogenic differentiation of bone marrow stromal cells (BMSC) and reversed APN-induced expression of the lysine-specific demethylases involved in regulating their commitment to the osteoblastic lineage. In conclusion, we found that APN regulates bone metabolism via central and peripheral mechanisms to decrease sympathetic tone, inhibit osteoclastic differentiation, and promote osteoblastic commitment of BMSC.
Assuntos
Adiponectina/farmacologia , Conservadores da Densidade Óssea/farmacologia , Medula Óssea/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Proteínas Recombinantes/farmacologia , Adiponectina/antagonistas & inibidores , Adiponectina/química , Adiponectina/genética , Adiposidade/efeitos dos fármacos , Animais , Conservadores da Densidade Óssea/administração & dosagem , Conservadores da Densidade Óssea/antagonistas & inibidores , Conservadores da Densidade Óssea/química , Medula Óssea/metabolismo , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/metabolismo , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Hipotálamo/citologia , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Infusões Intraventriculares , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Conformação Proteica , Radiografia , Distribuição Aleatória , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/químicaRESUMO
Multiple sclerosis (MS) is a presumed autoimmune disease directed against central nervous system (CNS) myelin, in which diet and obesity are implicated as risk factors. Immune responses can be influenced by molecules produced by fat cells, called adipokines. Adiponectin is an adipokine with anti-inflammatory effects. We tested the hypothesis that adiponectin has a protective role in the EAE model for MS, that can be induced by immunization with myelin antigens or transfer of myelin-specific T lymphocytes. Adiponectin deficient (ADPKO) mice developed worse EAE with greater CNS inflammation, demyelination, and axon injury. Lymphocytes from myelin-immunized ADPKO mice proliferated more, produced higher amounts of IFN-γ, IL-17, TNF-α, IL-6, and transferred more severe EAE than wild type (WT) lymphocytes. At EAE peak, the spleen and CNS of ADPKO had fewer regulatory T (Treg) cells than WT mice and during EAE recovery, Foxp3, IL-10 and TGF-ß expression levels in the CNS were reduced in ADPKO compared with WT mice. Treatment with globular adiponectin in vivo ameliorated EAE, and was associated with an increase in Treg cells. These data indicate that adiponectin is an important regulator of T-cell functions during EAE, suggesting a new avenue of investigation for MS treatment.
Assuntos
Adiponectina/fisiologia , Encefalomielite Autoimune Experimental/imunologia , Ativação Linfocitária , Linfócitos T Reguladores/imunologia , Adiponectina/administração & dosagem , Adiponectina/deficiência , Adiponectina/genética , Transferência Adotiva , Animais , Autoimunidade , Proliferação de Células , Células Cultivadas , Sistema Nervoso Central/imunologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/tratamento farmacológico , Fatores de Transcrição Forkhead/biossíntese , Interferon gama/biossíntese , Interleucina-10/biossíntese , Interleucina-17/biossíntese , Interleucina-6/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Esclerose Múltipla/imunologia , Bainha de Mielina/imunologia , Fatores de Risco , Baço/imunologia , Células Th1/imunologia , Células Th1/transplante , Fator de Crescimento Transformador beta/biossíntese , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Type 2 diabetes (T2D) is a widespread health condition both in the United States and around the world, with insulin resistance playing a critical role in its development. Effective treatment strategies are essential for managing T2D and mitigating associated risks. Adiponectin (APN), secreted by adipocytes, exhibits an inverse correlation with obesity-related adiposity, and its levels are negatively associated with insulin resistance and body mass index. This study aimed to enhance endogenous APN levels in a diet-induced obese (DIO) mouse model using lipid nanoparticles (LNP) as safe delivery agents for APN mRNA conjugates. The results indicate that APN-mRNA-LNP administration successfully induced APN synthesis in various tissues, including muscle, liver, kidney, pancreas, and adipose cells. This induction was associated with several positive outcomes, such as preventing diet-induced body weight gain, improving hyperglycemia by promoting Glut-4 expression, alleviating diabetic nephropathy symptoms by blocking the EGFR pathway, and reducing pro-inflammatory cytokine production. In addition, the treatment demonstrated enhanced insulin sensitivity by activating DGKd and inhibiting PKCε. This resulted in reactivation of insulin receptors in insulin target tissues and stimulation of insulin secretion from pancreatic beta cells. The findings of the present study highlight the potential of APN-mRNA-LNP-based nucleic acid therapy as a treatment for type 2 diabetes, offering a comprehensive approach to addressing its complexities.
RESUMO
AIMS/HYPOTHESIS: Adaptor protein, phosphotyrosine interaction, pleckstrin homology domain and leucine zipper containing 1 (APPL1) is an adapter protein that positively mediates adiponectin signalling. Deficiency of APPL1 in the target tissues of insulin induces insulin resistance. We therefore aimed, in the present study, to determine its role in regulating pancreatic beta cell function. METHODS: A hyperglycaemic clamp test was performed to determine insulin secretion in APPL1 knockout (KO) mice. Glucose- and adiponectin-induced insulin release was measured in islets from APPL1 KO mice or INS-1(832/13) cells with either APPL1 knockdown or overproduction. RT-PCR and western blotting were conducted to analyse gene expression and protein abundance. Oxygen consumption rate (OCR), ATP production and mitochondrial membrane potential were assayed to evaluate mitochondrial function. RESULTS: APPL1 is highly expressed in pancreatic islets, but its levels are decreased in mice fed a high-fat diet and db/db mice compared with controls. Deletion of the Appl1 gene leads to impairment of both the first and second phases of insulin secretion during hyperglycaemic clamp tests. In addition, glucose-stimulated insulin secretion (GSIS) is significantly decreased in islets from APPL1 KO mice. Conversely, overproduction of APPL1 leads to an increase in GSIS in beta cells. In addition, expression levels of several genes involved in insulin production, mitochondrial biogenesis and mitochondrial OCR, ATP production and mitochondrial membrane potential are reduced significantly in APPL1-knockdown beta cells. Moreover, suppression or overexproduction of APPL1 inhibits or stimulates adiponectin-potentiated GSIS in beta cells, respectively. CONCLUSIONS/INTERPRETATION: Our study demonstrates the roles of APPL1 in regulating GSIS and mitochondrial function in pancreatic beta cells, which implicates APPL1 as a therapeutic target in the treatment of type 2 diabetes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Glucose/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Mitocôndrias/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Animais , Western Blotting , Linhagem Celular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
APPL1 is an adaptor protein that plays a critical role in regulating adiponectin and insulin signaling. However, how APPL1 is regulated under normal and pathological conditions remains largely unknown. In this study, we show that APPL1 undergoes phosphorylation at Ser(430) and that this phosphorylation is enhanced in the liver of obese mice displaying insulin resistance. In cultured mouse hepatocytes, APPL1 phosphorylation at Ser(430) is stimulated by phorbol 12-myristate 13-acetate, an activator of classic PKC isoforms, and by the endoplasmic reticulum (ER) stress inducer, thapsigargin. Overexpression of wild-type but not dominant negative PKCα increases APPL1 phosphorylation at Ser(430) in mouse hepatocytes. In addition, suppressing PKCα expression by shRNA in hepatocytes reduces ER stress-induced APPL1 phosphorylation at Ser(430) as well as the inhibitory effect of ER stress on insulin-stimulated Akt phosphorylation. Consistent with a negative regulatory role of APPL1 phosphorylation at Ser(430) in insulin signaling, overexpression of APPL1(S430D) but not APPL1(S430A) impairs the potentiating effect of APPL1 on insulin-stimulated Akt phosphorylation at Thr(308). Taken together, our results identify APPL1 as a novel target in ER stress-induced insulin resistance and PKCα as the kinase mediating ER stress-induced phosphorylation of APPL1 at Ser(430).
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Estresse do Retículo Endoplasmático , Hepatócitos/metabolismo , Resistência à Insulina , Processamento de Proteína Pós-Traducional , Animais , Linhagem Celular , Ativadores de Enzimas/farmacologia , Hepatócitos/enzimologia , Humanos , Insulina/fisiologia , Isoenzimas/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/metabolismo , Fosforilação , Proteína Quinase C-alfa/metabolismo , Serina/metabolismo , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Adiponectin, also known as Acrp30, is an adipose tissue-derived hormone with anti-atherogenic, anti-diabetic and insulin sensitizing properties. Two seven-transmembrane domain-containing proteins, AdipoR1 and AdipoR2, have recently been identified as adiponectin receptors, yet signalling events downstream of these receptors remain poorly defined. By using the cytoplasmic domain of AdipoR1 as bait, we screened a yeast two-hybrid cDNA library derived from human fetal brain. This screening led to the identification of a phosphotyrosine binding domain and a pleckstrin homology domain-containing adaptor protein, APPL1 (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding (PTB) domain and leucine zipper motif). APPL1 interacts with adiponectin receptors in mammalian cells and the interaction is stimulated by adiponectin. Overexpression of APPL1 increases, and suppression of APPL1 level reduces, adiponectin signalling and adiponectin-mediated downstream events (such as lipid oxidation, glucose uptake and the membrane translocation of glucose transport 4 (GLUT4)). Adiponectin stimulates the interaction between APPL1 and Rab5 (a small GTPase) interaction, leading to increased GLUT4 membrane translocation. APPL1 also acts as a critical regulator of the crosstalk between adiponectin signalling and insulin signalling pathways. These results demonstrate a key function for APPL1 in adiponectin signalling and provide a molecular mechanism for the insulin sensitizing function of adiponectin.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adiponectina/metabolismo , Proteínas de Transporte/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Adiponectina/farmacologia , Animais , Células CHO , Proteínas de Transporte/química , Células Cultivadas , Cricetinae , Cricetulus , Perfilação da Expressão Gênica , Glucose/metabolismo , Humanos , Insulina/farmacologia , Camundongos , Dados de Sequência Molecular , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Ligação Proteica , Receptores de Adiponectina , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
Adiponectin is an adipokine playing an important role in regulating energy homeostasis and insulin sensitivity. However, the effect of adiponectin on bone metabolism shows contradictory results according to different research studies. In this study femurs were isolated from genetically double-labeled mBSP9.0Luc/ß-ACT-EGFP transgenic mice and were transplanted into adiponectin knock-out mice or wild type mice to investigate the effect of temporary exposure to adiponectin deficiency on bone growth and metabolism. We found that the growth of bone explants in adiponectin knock-out mice was significantly retarded. Histological analysis, microcomputed tomography analysis, and tartrate-resistant acid phosphatase staining revealed reduced trabecular bone volume, decreased cortical bone, and increased osteoclast number in bone explants in adiponectin knock-out mice. We then found that adiponectin inhibits RANKL-induced osteoclastogenesis from RAW264.7 cells and down-regulates RANKL-enhanced expressions of osteoclastogenic regulators including NFAT2, TRAF6, cathepsin K, and tartrate-resistant acid phosphatase. Adiponectin also increases osteoclast apoptosis and decreases survival/proliferation of osteoclast precursor cells. Using siRNA specifically targeting APPL1, the first identified adaptor protein of adiponectin signaling, we found that the inhibitory effect of adiponectin on osteoclasts was induced by APPL1-mediated down-regulation of Akt1 activity. In addition, overexpression of Akt1 successfully reversed adiponectin-induced inhibition in RANKL-stimulated osteoclast differentiation. In conclusion, adiponectin is important in maintaining the balance of energy metabolism, inflammatory responses, and bone formation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adiponectina/metabolismo , Reabsorção Óssea/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adiponectina/genética , Adiponectina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Western Blotting , Reabsorção Óssea/genética , Catepsina K/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Fêmur/diagnóstico por imagem , Fêmur/transplante , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Osteoclastos/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-akt/genética , Ligante RANK/farmacologia , Radiografia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The natural polyphenol resveratrol (RSV) displays a wide spectrum of health beneficial activities, yet the precise mechanisms remain to be fully elucidated. Here we show that RSV promotes the multimerization and cellular levels of adiponectin in 3T3-L1 adipocytes. The stimulatory effect of RSV was not affected by knocking out Sirt1, but was diminished by suppressing the expression levels of DsbA-L, a recently identified adiponectin-interactive protein that promotes adiponectin multimerization. Suppression of the Akt signaling pathway resulted in an increase in the expression levels of DsbA-L and adiponectin. On the other hand, knocking out FOXO1 or suppressing the activity or expression levels of the AMP-activated protein kinase (AMPK) down-regulated DsbA-L and adiponectin. The stimulatory effect of RSV on adiponectin and DsbA-L expression was completely diminished in FOXO1-suppressed and AMPK-inactivated 3T3-L1 adipocytes. Taken together, our results demonstrate that RSV promotes adiponectin multimerization in 3T3-L1 adipocytes via a Sirt1-independent mechanism. In addition, we show that the stimulatory effect of RSV is regulated by both the Akt/FOXO1 and the AMPK signaling pathways. Last, we show that DsbA-L plays a critical role in the promoting effect of RSV on adiponectin multimerization and cellular levels.
Assuntos
Adiponectina/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Glutationa Transferase/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estilbenos/farmacologia , Regulação para Cima/efeitos dos fármacos , Células 3T3-L1 , Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adiponectina/química , Adiponectina/genética , Animais , Proteína Forkhead Box O1 , Glutationa Transferase/deficiência , Glutationa Transferase/genética , Camundongos , Multimerização Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , ResveratrolRESUMO
Resveratrol (RSV) is a naturally occurring polyphenol that has been found to exert antioxidant, anti-inflammatory, and neuroprotective properties. However, how RSV exerts its beneficial health effects remains largely unknown. Here, we show that RSV inhibits insulin- and leucine-stimulated mTOR signaling in C2C12 fibroblasts via a Sirt1-independent mechanism. Treating C2C12 cells with RSV dramatically inhibited insulin-stimulated Akt, S6 kinase, and 4E-BP1 phosphorylation but had little effect on tyrosine phosphorylation of the insulin receptor and activation of the p44/42 MAPK signaling pathway. RSV treatment also partially blocked mTOR and S6 kinase phosphorylation in TSC1/2-deficient mouse embryonic fibroblasts, suggesting the presence of an inhibitory site downstream of TSC1/2. Knocking out PDK1 or suppressing AMP-activated protein kinase had little effect on leucine-stimulated mTOR signaling. On the other hand, RSV significantly increased the association between mTOR and its inhibitor, DEPTOR. Furthermore, the inhibitory effect of RSV on leucine-stimulated mTOR signaling was greatly reduced in cells in which the expression levels of DEPTOR were suppressed by RNAi. Taken together, our studies reveal that RSV inhibits leucine-stimulated mTORC1 activation by promoting mTOR/DEPTOR interaction and thus uncover a novel mechanism by which RSV negatively regulates mTOR activity.