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AIMS/HYPOTHESIS: In the postprandial state, the liver regulates glucose homeostasis by glucose uptake and conversion to glycogen and lipids. Glucose and insulin signalling finely regulate glycogen synthesis through several mechanisms. Glucose uptake in hepatocytes is favoured by the insulin receptor isoform A (IRA), rather than isoform B (IRB). Thus, we hypothesised that, in hepatocytes, IRA would increase glycogen synthesis by promoting glucose uptake and glycogen storage. METHODS: We addressed the role of insulin receptor isoforms on glycogen metabolism in vitro in immortalised neonatal hepatocytes. In vivo, IRA or IRB were specifically expressed in the liver using adeno-associated virus vectors in inducible liver insulin receptor knockout (iLIRKO) mice, a model of type 2 diabetes. The role of IR isoforms in glycogen synthesis and storage in iLIRKO was subsequently investigated. RESULTS: In immortalised hepatocytes, IRA, but not IRB expression induced an increase in insulin signalling that was associated with elevated glycogen synthesis, glycogen synthase activity and glycogen storage. Similarly, elevated IRA, but not IRB expression in the livers of iLIRKO mice induced an increase in glycogen content. CONCLUSIONS/INTERPRETATION: We provide new insight into the role of IRA in the regulation of glycogen metabolism in cultured hepatocytes and in the livers of a mouse model of type 2 diabetes. Our data strongly suggest that IRA is more efficient than IRB at promoting glycogen synthesis and storage. Therefore, we suggest that IRA expression in the liver could provide an interesting therapeutic approach for the regulation of hepatic glucose content and glycogen storage.
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Diabetes Mellitus Tipo 2/metabolismo , Glicogênio Fosforilase/metabolismo , Glicogênio Sintase/metabolismo , Glicogênio/metabolismo , Glicogênio Hepático/metabolismo , Fígado/metabolismo , Isoformas de Proteínas/metabolismo , Receptor de Insulina/metabolismo , Animais , Western Blotting , Linhagem Celular , Diabetes Mellitus Tipo 2/genética , Glucose/metabolismo , Glicogênio Fosforilase/genética , Glicogênio Sintase/genética , Glicogenólise , Hepatócitos , Camundongos , Camundongos Knockout , Isoformas de Proteínas/genética , Receptor de Insulina/genéticaRESUMO
BACKGROUND: Several translational studies have identified the differential role between saturated and unsaturated fatty acids at cardiovascular level. However, the molecular mechanisms that support the protective role of oleate in cardiovascular cells are poorly known. For these reasons, we studied the protective role of oleate in the insulin resistance and in the atherosclerotic process at cellular level such as in cardiomyocytes (CMs), vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). METHODS: The effect of oleate in the cardiovascular insulin resistance, vascular dysfunction, inflammation, proliferation and apoptosis of VSMCs were analyzed by Western blot, qRT-PCR, BrdU incorporation and cell cycle analysis. RESULTS: Palmitate induced insulin resistance. However, oleate not only did not induce cardiovascular insulin resistance but also had a protective effect against insulin resistance induced by palmitate or TNFα. One mechanism involved might be the prevention by oleate of JNK-1/2 or NF-κB activation in response to TNF-α or palmitate. Oleate reduced MCP-1 and ICAM-1 and increased eNOS expression induced by proinflammatory cytokines in ECs. Furthermore, oleate impaired the proliferation induced by TNF-α, angiotensin II or palmitate and the apoptosis induced by TNF-α or thapsigargin in VSMCs. CONCLUSIONS: Our data suggest a differential role between oleate and palmitate and support the concept of the cardioprotector role of oleate as the main lipid component of virgin olive oil. Thus, oleate protects against cardiovascular insulin resistance, improves endothelial dysfunction in response to proinflammatory signals and finally, reduces proliferation and apoptosis in VSMCs that may contribute to an ameliorated atherosclerotic process and plaque stability.
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Aterosclerose/metabolismo , Resistência à Insulina , Músculo Liso Vascular/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Ácido Oleico/farmacologia , RNA Mensageiro/efeitos dos fármacos , Angiotensina II/farmacologia , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Quimiocina CCL2/efeitos dos fármacos , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Inflamação , Molécula 1 de Adesão Intercelular/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Músculo Liso Vascular/citologia , Miócitos Cardíacos/metabolismo , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo III/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Palmitatos/farmacologia , Ácido Palmítico/farmacologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/farmacologia , Vasoconstritores/farmacologiaRESUMO
BACKGROUND: It has been reported that increased expression of UCP-2 in the vasculature may prevent the development of atherosclerosis in patients with increased production of reactive oxygen species, as in the diabetes, obesity or hypertension. Thus, a greater understanding in the modulation of UCP-2 could improve the atherosclerotic process. However, the effect of TNF-α or insulin modulating UCP-2 in the vascular wall is completely unknown. In this context, we propose to study new molecular mechanisms that help to explain whether the moderate hyperinsulinemia or lowering TNF-α levels might have a protective role against vascular damage mediated by UCP-2 expression levels. METHODS: We analyzed the effect of insulin or oleic acid in presence or not of TNF-α on UCP-2 expression in murine endothelial and vascular smooth muscle cells. At this step, we wondered if some mechanisms studied in vitro could be of any relevance in vivo. We used the following experimental models: ApoE-/- mice under Western type diet for 2, 6, 12 or 18 weeks, BATIRKO mice under high-fat diet for 16 weeks and 52-week-old BATIRKO mice with o without anti-TNF-α antibody pre-treatment. RESULTS: Firstly, we found that TNF-α pre-treatment reduced UCP-2 expression induced by insulin in vascular cells. Secondly, we observed a progressive reduction of UCP-2 levels together with an increase of lipid depots and lesion area in aorta from ApoE-/- mice. In vivo, we also observed that moderate hyperinsulinemic obese BATIRKO mice have lower TNF-α and ROS levels and increased UCP-2 expression levels within the aorta, lower lipid accumulation, vascular dysfunction and macrovascular damage. We also observed that the anti-TNF-α antibody pre-treatment impaired the loss of UCP-2 expression within the aorta and relieved vascular damage observed in 52-week-old BATIRKO mice. Finally, we observed that the pretreatment with iNOS inhibitor prevented UCP-2 reduction induced by TNF-α in vascular cells. Moreover, iNOS levels are augmented in aorta from mice with lower UCP-2 levels and higher TNF-α levels. CONCLUSIONS: Our data suggest that moderate hyperinsulinemia in response to insulin resistance or lowering of TNF-α levels within the aorta attenuates vascular damage, this protective effect being mediated by UCP-2 expression levels through iNOS.
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Insulina/farmacologia , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/biossíntese , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/biossíntese , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Células Cultivadas , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Knockout , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Proteína Desacopladora 2RESUMO
Patients with immune-mediated inflammatory diseases (IMIDs), such as rheumatoid arthritis and inflammatory bowel disease, are at increased risk of infection. International guidelines recommend vaccination to limit this risk of infection, although live attenuated vaccines are contraindicated once immunosuppressive therapy has begun. Biologic therapies used to treat IMIDs target the immune system to stop chronic pathogenic process but may also attenuate the protective immune response to vaccines. Here, we review the current knowledge regarding vaccine responses in IMID patients receiving treatment with biologic therapies, with a focus on the interleukin (IL)-12/23 inhibitors. B cell-depleting therapies, such as rituximab, strongly impair vaccines immunogenicity, and tumor necrosis factor (TNF) inhibitors and the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) fusion protein abatacept are also associated with attenuated antibody responses, which are further diminished in patients taking concomitant immunosuppressants. On the other hand, integrin, IL-6, IL-12/23, IL-17, and B-cell activating factor (BAFF) inhibitors do not appear to affect the immune response to several vaccines evaluated. Importantly, treatment with biologic therapies in IMID patients is not associated with an increased risk of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or developing severe disease. However, the efficacy of SARS-CoV-2 vaccines on IMID patients may be reduced compared with healthy individuals. The impact of biologic therapies on the response to SARS-CoV-2 vaccines seems to replicate what has been described for other vaccines. SARS-CoV-2 vaccination appears to be safe and well tolerated in IMID patients. Attenuated but, in general, still protective responses to SARS-CoV-2 vaccination in the context of certain therapies warrant current recommendations for a third primary dose in IMID patients treated with immunosuppressive drugs.
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Phytoestrogens can have a neuroprotective effect towards ischemia-reperfusion-induced neuronal damage. However, their mechanism of action has not been well described. In this work, we investigate the type of neuronal cell death induced by oxygen and glucose deprivation (OGD) and resupply (OGDR) and pinpoint some of the signaling mechanisms whereby the neuroprotective effects of phytoestrogens occur in these conditions. First, we found that autophagy initiation affords neuronal protection upon neuronal damage induced by OGD and OGDR. The mammalian target of rapamycin/ribosomal S6 kinase (mTOR/S6K) pathway is blocked in these conditions, and we provide evidence that this is mediated by modulation of both the 5' AMP-activated protein kinase (AMPK) and phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) pathways. These are dampened up or down, respectively, under OGDR-induced neuronal damage. In contrast, the MAPK-Erk kinase/extracellular signal-regulated kinase (MEK/ERK) pathway is increased under these conditions. Regarding the pathways affected by phytoestrogens, we show that their protective properties require autophagy initiation, but at later stages, they decrease mitogen-activated protein kinase (MAPK) and AMPK activation and increase mTOR/S6K activation. Collectively, our results put forward a novel mode of action where phytoestrogens play a dual role in the regulation of autophagy by acting as autophagy initiation enhancers when autophagy is a neuroprotective and pro-survival mechanism, and as autophagy initiation inhibitors when autophagy is a pro-death mechanism. Finally, our results support the therapeutic potential of phytoestrogens in brain ischemia by modulating autophagy.
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Among the main complications associated with obesity are insulin resistance and altered glucose and lipid metabolism within the liver. It has previously been described that insulin receptor isoform A (IRA) favors glucose uptake and glycogen storage in hepatocytes compared with isoform B (IRB), improving glucose homeostasis in mice lacking liver insulin receptor. Thus, we hypothesized that IRA could also improve glucose and lipid metabolism in a mouse model of high-fat-diet-induced obesity. We addressed the role of insulin receptor isoforms in glucose and lipid metabolism in vivo We expressed IRA or IRB specifically in the liver by using adeno-associated viruses (AAVs) in a mouse model of diet-induced insulin resistance and obesity. IRA, but not IRB, expression induced increased glucose uptake in the liver and muscle, improving insulin tolerance. Regarding lipid metabolism, we found that AAV-mediated IRA expression also ameliorated hepatic steatosis by decreasing the expression of Fasn, Pgc1a, Acaca and Dgat2 and increasing Scd-1 expression. Taken together, our results further unravel the role of insulin receptor isoforms in hepatic glucose and lipid metabolism in an insulin-resistant scenario. Our data strongly suggest that IRA is more efficient than IRB at favoring hepatic glucose uptake, improving insulin tolerance and ameliorating hepatic steatosis. Therefore, we conclude that a gene therapy approach for hepatic IRA expression could be a safe and promising tool for the regulation of hepatic glucose consumption and lipid metabolism, two key processes in the development of non-alcoholic fatty liver disease associated with obesity.This article has an associated First Person interview with the first author of the paper.
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Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Glucose/metabolismo , Fígado/metabolismo , Obesidade/metabolismo , Receptor de Insulina/metabolismo , Animais , Dependovirus/metabolismo , Dieta Hiperlipídica , Modelos Animais de Doenças , Regulação da Expressão Gênica , Resistência à Insulina , Secreção de Insulina , Metabolismo dos Lipídeos/genética , Masculino , Camundongos Endogâmicos C57BL , Músculos/metabolismo , Especificidade de Órgãos , Isoformas de Proteínas/metabolismoRESUMO
This review focuses on the contribution of white, brown, and perivascular adipose tissues to the pathophysiology of obesity and its associated metabolic and vascular complications. Weight gain in obesity generates excess of fat, usually visceral fat, and activates the inflammatory response in the adipocytes and then in other tissues such as liver. Therefore, low systemic inflammation responsible for insulin resistance contributes to atherosclerotic process. Furthermore, an inverse relationship between body mass index and brown adipose tissue activity has been described. For these reasons, in recent years, in order to combat obesity and its related complications, as a complement to conventional treatments, a new insight is focusing on the role of the thermogenic function of brown and perivascular adipose tissues as a promising therapy in humans. These lines of knowledge are focused on the design of new drugs, or other approaches, in order to increase the mass and/or activity of brown adipose tissue or the browning process of beige cells from white adipose tissue. These new treatments may contribute not only to reduce obesity but also to prevent highly prevalent complications such as type 2 diabetes and other vascular alterations, such as hypertension or atherosclerosis.
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Obesity is one of the major risk factors for the development of cardiovascular diseases and is characterized by abnormal accumulation of adipose tissue, including perivascular adipose tissue (PVAT). However, brown adipose tissue (BAT) activation reduces visceral adiposity. To demonstrate that severe brown fat lipoatrophy might accelerate atherosclerotic process, we generated a new mouse model without insulin receptor (IR) in BAT and without apolipoprotein (Apo)E (BAT-specific IR knockout [BATIRKO];ApoE(-/-) mice) and assessed vascular and metabolic alterations associated to obesity. In addition, we analyzed the contribution of the adipose organ to vascular inflammation. Brown fat lipoatrophy induces visceral adiposity, mainly in gonadal depot (gonadal white adipose tissue [gWAT]), severe glucose intolerance, high postprandial glucose levels, and a severe defect in acute insulin secretion. BATIRKO;ApoE(-/-) mice showed greater hypertriglyceridemia than the obtained in ApoE(-/-) and hypercholesterolemia similar to ApoE(-/-) mice. BATIRKO;ApoE(-/-) mice, in addition to primary insulin resistance in BAT, also showed a significant decrease in insulin signaling in liver, gWAT, heart, aorta artery, and thoracic PVAT. More importantly, our results suggest that severe brown fat lipoatrophy aggravates the atherosclerotic process, characterized by a significant increase of lipid depots, atherosclerotic coverage, lesion size and complexity, increased macrophage infiltration, and proinflammatory markers expression. Finally, an increase of TNF-α and leptin as well as a decrease of adiponectin by BAT, gWAT, and thoracic PVAT might also be responsible of vascular damage. Our results suggest that severe brown lipoatrophy aggravates atherosclerotic process. Thus, BAT activation might protect against obesity and its associated metabolic alterations.
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Tecido Adiposo Marrom/metabolismo , Aterosclerose/etiologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Lipoatrófica/complicações , Adiponectina/metabolismo , Tecido Adiposo Branco/metabolismo , Adiposidade , Animais , Apolipoproteínas E/genética , Biomarcadores/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Lipoatrófica/metabolismo , Glucose/metabolismo , Resistência à Insulina , Leptina/metabolismo , Metabolismo dos Lipídeos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Insulina/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Type 2 diabetes mellitus is a complex metabolic disease and its pathogenesis involves abnormalities in both peripheral insulin action and insulin secretion. Previous in vitro data showed that insulin receptor isoform A, but not B, favours basal glucose uptake through its specific association with endogenous GLUT1/2 in murine hepatocytes and beta cells. With this background, we hypothesized that hepatic expression of insulin receptor isoform A in a mouse model of type 2 diabetes could potentially increase the glucose uptake of these cells, decreasing the hyperglycaemia and therefore ameliorating the diabetic phenotype. To assure this hypothesis, we have developed recombinant adeno-associated viral vectors expressing insulin receptor isoform A (IRA) or isoform B (IRB) under the control of a hepatocyte--specific promoter. Our results demonstrate that in the long term, hepatic expression of IRA in diabetic mice is more efficient than IRB in ameliorating glucose intolerance. Consequently, it impairs the induction of compensatory mechanisms through beta cell hyperplasia and/or hypertrophy that finally lead to beta cell failure, reverting the diabetic phenotype in about 8â weeks. Our data suggest that long-term hepatic expression of IRA could be a promising therapeutic approach for the treatment of type 2 diabetes mellitus.
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Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Intolerância à Glucose/metabolismo , Receptor de Insulina/metabolismo , Animais , Proliferação de Células , Dependovirus/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/patologia , Glucose/metabolismo , Intolerância à Glucose/patologia , Proteínas de Fluorescência Verde/metabolismo , Homeostase , Hiperplasia , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Fígado/metabolismo , Camundongos Knockout , Isoformas de Proteínas/metabolismoRESUMO
The main compensatory response to insulin resistance is the pancreatic beta cell hyperplasia to account for increased insulin secretion. In fact, in a previous work we proposed a liver-pancreas endocrine axis with IGF-I (insulin-like growth factor type I) secreted by the liver acting on IRA insulin receptor in beta cells from iLIRKO mice (inducible Liver Insulin Receptor KnockOut) that showed a high IRA/IRB ratio. However, the role of insulin receptor isoforms in the IGF-I-induced beta cell proliferation as well as the underlying molecular mechanisms remain poorly understood. For this purpose, we have used four immortalized mouse beta cell lines: bearing IR (IRLoxP), lacking IR (IRKO), expressing exclusively IRA (IRA), or alternatively expressing IRB (IRB). Pancreatic beta cell proliferation studies showed that IRA cells are more sensitive than those expressing IRB to the mitogenic response induced by IGF-I, acting through the pathway IRA/IRS-1/2/αp85/Akt/mTORC1/p70S6K. More importantly, IRA beta cells, but not IRB, showed an increased glucose uptake as compared with IRLoxP cells, this effect being likely owing to an enhanced association between Glut-1 and Glut-2 with IRA. Overall, our results strongly suggest a prevalent role of IRA in glucose availability and IGF-I-induced beta cell proliferation mainly through mTORC1. These results could explain, at least partially, the role played by the liver-secreted IGF-I in the compensatory beta cell hyperplasia observed in response to severe hepatic insulin resistance in iLIRKO mice.
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Glucose/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Células Secretoras de Insulina/fisiologia , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Complexos Multiproteicos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
The action of (Z)-N-(2-bromo-5-hydroxy-4-methoxybenzylidene)-2-methylpropan-2-amine oxide (RP6) on rat cortical neurons in culture, under oxygen-glucose-deprivation conditions, is reported. Cortical neurons in culture were treated during 1 h with OGD. After, they were placed under normal conditions during 24 h (reperfusion) in absence and presence of RP6. Different parameters were measured under each condition (control, 1 h OGD and 1 h OGD + reperfusion in absence and presence of RP6). RP6 protects neurons against ROS generation, lipid peroxidation levels, LDH release and mitochondrial membrane potential alteration, when administered during reperfusion after the OGD damage. Consequently, these results show that nitrone RP6 protects cells against ischemia injury produced during the reoxygenation, and could be a potential drug for the ictus therapy.