RESUMO
Rationale: MG53's known function in facilitating tissue repair and anti-inflammation has broad applications to regenerative medicine. There is controversy regarding MG53's role in the development of type 2 diabetes mellitus. Objective: This study aims to address this controversy - whether MG53's myokine function contributes to inhibition of insulin signaling in muscle, heart, and liver tissues. Study design: We determined the binding affinity of the recombinant human MG53 (rhMG53) to the insulin receptor extracellular domain (IR-ECD) and found low affinity of interaction with Kd (>480 nM). Using cultured C2C12 myotubes and HepG2 cells, we found no effect of rhMG53 on insulin-stimulated Akt phosphorylation (p-Akt). We performed in vivo assay with C57BL/6J mice subjected to insulin stimulation (1 U/kg, intraperitoneal injection) and observed no effect of rhMG53 on insulin-stimulated p-Akt in muscle, heart and liver tissues. Conclusion: Overall, our data suggest that rhMG53 can bind to the IR-ECD, however has a low likelihood of a physiologic role, as the Kd for binding is ~10,000 higher than the physiologic level of MG53 present in the serum of rodents and humans (~10 pM). Our findings question the notion proposed by Xiao and colleagues - whether targeting circulating MG53 opens a new therapeutic avenue for type 2 diabetes mellitus and its complications.
Assuntos
Insulina , Fígado , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt , Receptor de Insulina , Animais , Humanos , Camundongos , Fosforilação/efeitos dos fármacos , Receptor de Insulina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fígado/metabolismo , Fígado/efeitos dos fármacos , Insulina/metabolismo , Insulina/farmacologia , Miocárdio/metabolismo , Células Hep G2 , Músculo Esquelético/metabolismo , Músculo Esquelético/efeitos dos fármacos , Masculino , Transdução de Sinais/efeitos dos fármacos , Diabetes Mellitus Tipo 2/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Citocinas/metabolismo , Proteínas de MembranaRESUMO
Restenosis following percutaneous revascularization is a major challenge in patients with insulin resistance and diabetes. Currently, the vascular effects of insulin are not fully understood. In vitro, insulin's effects on endothelial cells (ECs) are beneficial, whereas on vascular smooth muscle cells (SMCs), they are mitogenic. We previously demonstrated a suppressive effect of insulin on neointimal growth under insulin-sensitive conditions that was abolished in insulin-resistant conditions. Here, we aimed to determine the cell-specific effects of insulin on neointimal growth in a model of restenosis under insulin-sensitive and insulin-resistant conditions. Vascular cell-specific insulin receptor (IR)-deficient mice were fed a low-fat diet (LFD) or a high-fat, high-sucrose diet (HFSD) and implanted with an insulin pellet or vehicle prior to femoral artery wire injury. In insulin-sensitive conditions, insulin decreased neointimal growth only in controls. However, under insulin-resistant conditions, insulin had no effect in either control, EC-specific or SMC-specific IR-deficient mice. These data demonstrate that EC and SMC IRs are required for the anti-restenotic effect of insulin in insulin-sensitive conditions and that, in insulin resistance, insulin has no adverse effect on vascular SMCs in vivo.
Assuntos
Modelos Animais de Doenças , Células Endoteliais , Resistência à Insulina , Insulina , Receptor de Insulina , Animais , Insulina/metabolismo , Insulina/farmacologia , Camundongos , Receptor de Insulina/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Neointima/patologia , Neointima/metabolismo , Masculino , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Camundongos Endogâmicos C57BLRESUMO
Dendrite pathology and synaptic loss result in neural circuit dysfunction, a common feature of neurodegenerative diseases. There is a lack of strategies that target dendritic and synaptic regeneration to promote neurorecovery. We show that daily human recombinant insulin eye drops stimulate retinal ganglion cell (RGC) dendrite and synapse regeneration during ocular hypertension, a risk factor to develop glaucoma. We demonstrate that the ribosomal protein p70S6 kinase (S6K) is essential for insulin-dependent dendritic regrowth. Furthermore, S6K phosphorylation of the stress-activated protein kinase-interacting protein 1 (SIN1), a link between the mammalian target of rapamycin complexes 1 and 2 (mTORC1/2), is required for insulin-induced dendritic regeneration. Using two-photon microscopy live retinal imaging, we show that insulin rescues single-RGC light-evoked calcium (Ca2+) dynamics. We further demonstrate that insulin enhances neuronal survival and retina-brain connectivity leading to improved optomotor reflex-elicited behaviors. Our data support that insulin is a compelling pro-regenerative strategy with potential clinical implications for the treatment and management of glaucoma.
Assuntos
Glaucoma , Insulina , Células Ganglionares da Retina , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/efeitos dos fármacos , Glaucoma/tratamento farmacológico , Glaucoma/metabolismo , Glaucoma/patologia , Insulina/metabolismo , Insulina/farmacologia , Animais , Humanos , Camundongos , Modelos Animais de Doenças , Dendritos/metabolismo , Dendritos/efeitos dos fármacos , Sinapses/metabolismo , Sinapses/efeitos dos fármacos , Cálcio/metabolismoRESUMO
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a cell model now widely used to investigate pathophysiological features of cardiac tissue. Given the invaluable contribution hiPSC-CM could make for studies on cardio-metabolic disorders by defining a postnatal metabolic phenotype, our work herein focused on monitoring the insulin response in CM derived from the hiPSC line UKBi015-B. Western blot analysis on total cell lysates obtained from hiPSC-CM showed increased phosphorylation of both AKT and AS160 following insulin treatment, but failed to highlight any changes in the expression dynamics of the glucose transporter GLUT4. By contrast, the Western blot analysis of membrane fractions, rather than total lysates, revealed insulin-induced plasma membrane translocation of GLUT4, which is known to also occur in postnatal CM. Thus, these findings suggest that hiPSC-derived CMs exhibit an insulin response reminiscent to that of adult CMs regarding intracellular signaling and GLUT4 translocation to the plasma membrane, representing a suitable cellular model in the cardio-metabolic research field. Moreover, our studies also demonstrate the relevance of analyzing membrane fractions rather than total lysates in order to monitor GLUT4 dynamics in response to metabolic regulators in hiPSC-CMs.
Assuntos
Membrana Celular , Transportador de Glucose Tipo 4 , Células-Tronco Pluripotentes Induzidas , Insulina , Miócitos Cardíacos , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Transportador de Glucose Tipo 4/metabolismo , Miócitos Cardíacos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Insulina/metabolismo , Insulina/farmacologia , Membrana Celular/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosforilação , Diferenciação Celular , Proteínas Ativadoras de GTPase/metabolismo , Linhagem CelularRESUMO
With the development of Type 1 diabetes mellitus (T1DM), various complications can be caused. Hyperglycemia affects the microenvironment of cardiomyocytes, changes endoplasmic reticulum homeostasis, triggers unfolding protein response and eventually promotes myocardial apoptosis. However, insulin therapy alone cannot effectively combat the complications caused by T1DM. Forty adult beagles were randomly divided into five groups: control group, diabetes mellitus group, insulin group, insulin combined with NAC group, and NAC group. 24-hour blood glucose, 120-day blood glucose, 120-day body weight, and serum FMN content were observed, furthermore, hematoxylin-eosin staining, Periodic acid Schiff reagent staining, and Sirius red staining of the myocardium were evaluated. The protein expressions of GRP78, ATF6, IRE1, PERK, JNK, CHOP, caspase 3, Bcl2, and Bax were detected. Results of the pathological section of myocardial tissue indicated that insulin combined with NAC therapy could improve myocardial pathological injury and glycogen deposition. Additionally, insulin combined with NAC therapy down-regulates the expression of GRP78, ATF6, IRE1, PERK, JNK, CHOP, caspase3, and Bax. These findings suggest that NAC has a phylactic effect on myocardial injury in beagles with T1DM, and the mechanism may be related to the improvement of endoplasmic reticulum stress-induced apoptosis.
Assuntos
Acetilcisteína , Diabetes Mellitus Tipo 1 , Retículo Endoplasmático , Insulina , Miocárdio , Animais , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/complicações , Insulina/farmacologia , Insulina/metabolismo , Cães , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Acetilcisteína/farmacologia , Miocárdio/metabolismo , Miocárdio/patologia , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Masculino , Transdução de Sinais/efeitos dos fármacosRESUMO
Women with a history of gestational diabetes mellitus (GDM) have a significantly greater lifetime risk of developing cardiovascular disease and type 2 diabetes compared with women who had an uncomplicated pregnancy (HC). Microvascular endothelial dysfunction, mediated via reduced nitric oxide (NO)-dependent dilation secondary to increases in oxidative stress, persists after pregnancy complicated by GDM. We examined whether this microvascular dysfunction reduces insulin-mediated vascular responses in women with a history of GDM. We assessed in vivo microvascular endothelium-dependent vasodilator function by measuring cutaneous vascular conductance responses to graded infusions of acetylcholine (10-10-10-1 M) and insulin (10-8-10-4 M) in control sites and sites treated with 15 mM l-NAME [NG-nitro-l-arginine methyl ester; NO-synthase (NOS) inhibitor] or 5 mM l-ascorbate. We also measured protein expression of total endothelial NOS (eNOS), insulin-mediated eNOS phosphorylation, and endothelial nitrotyrosine in isolated endothelial cells from GDM and HC. Women with a history of GDM had reduced acetylcholine (P < 0.001)- and insulin (P < 0.001)-mediated dilation, and the NO-dependent responses to both acetylcholine (P = 0.006) and insulin (P = 0.006) were reduced in GDM compared with HC. Insulin stimulation increased phosphorylated eNOS content in HC (P = 0.009) but had no effect in GDM (P = 0.306). Ascorbate treatment increased acetylcholine (P < 0.001)- and insulin (P < 0.001)-mediated dilation in GDM, and endothelial cell nitrotyrosine expression was higher in GDM compared with HC (P = 0.014). Women with a history of GDM have attenuated microvascular vasodilation responses to insulin, and this attenuation is mediated, in part, by reduced NO-dependent mechanisms. Our findings further implicate increased endothelial oxidative stress in this microvascular insulin resistance.NEW & NOTEWORTHY Women who have gestational diabetes during pregnancy are at a greater risk for cardiovascular disease and type 2 diabetes in the decade following pregnancy. The mechanisms mediating this increased risk are unclear. Herein, we demonstrate that insulin-dependent microvascular responses are reduced in women who had gestational diabetes, despite the remission of glucose intolerance. This reduced microvascular sensitivity to insulin may contribute to increased cardiovascular disease and type 2 diabetes risk in these women.
Assuntos
Diabetes Gestacional , Insulina , Microvasos , Óxido Nítrico Sintase Tipo III , Óxido Nítrico , Vasodilatação , Feminino , Diabetes Gestacional/fisiopatologia , Diabetes Gestacional/metabolismo , Humanos , Gravidez , Vasodilatação/efeitos dos fármacos , Insulina/farmacologia , Adulto , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Microvasos/metabolismo , Microvasos/efeitos dos fármacos , Microvasos/fisiopatologia , Acetilcolina/farmacologia , Vasodilatadores/farmacologia , Fosforilação , Estresse Oxidativo/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Endotélio Vascular/efeitos dos fármacos , Tirosina/análogos & derivados , Tirosina/metabolismo , Estudos de Casos e Controles , NG-Nitroarginina Metil Éster/farmacologia , Pele/irrigação sanguíneaRESUMO
BACKGROUND: Burns are classified according to their mechanism of injury, depth, affected body area, affected region or part of the body, and extent of the lesions. Topical insulin modulates the healing process. However, studies evaluating the effects of topical insulin treatment on burns in human patients are lacking. PURPOSE: The purpose of this study was to investigate the effects of topical insulin on healing time of second-degree burns. METHODS: In this nonrandomized clinical trial, patients with second-degree burns were allocated to a control group (CG) or an intervention group (IG) in which wounds were treated with 1% silver sulfadiazine and topical insulin, respectively. RESULTS: Healing time was significantly shorter in the IG relative to the CG (9.1 ± 1.9 days and 12.7 ± 3.3 days, respectively; P < .05). The estimated burn area was similar in both groups (CG 1.44 ± 1.0%; IG 1.42 ± 0.53%). CONCLUSION: In this study, topical insulin reduced healing time in second-degree burns. Further investigation is warranted to support wider use in clinical practice.
Assuntos
Administração Tópica , Queimaduras , Insulina , Cicatrização , Humanos , Queimaduras/tratamento farmacológico , Queimaduras/fisiopatologia , Cicatrização/efeitos dos fármacos , Insulina/uso terapêutico , Insulina/administração & dosagem , Insulina/farmacologia , Feminino , Masculino , Adulto , Pessoa de Meia-Idade , Sulfadiazina de Prata/uso terapêutico , Sulfadiazina de Prata/farmacologia , Sulfadiazina de Prata/administração & dosagem , Fatores de TempoRESUMO
Precision-cut liver slices (PCLS) are increasingly used as a model to investigate anti-fibrotic therapies. However, many studies use PCLS from healthy animals treated with pro-fibrotic stimuli in culture, which reflects only the early stages of fibrosis. The effects of different culture conditions on PCLS from cirrhotic animals has not been well characterized and there is no consensus on optimal methods. In this study, we report a method for the collection and culture of cirrhotic PCLS and compare the effect of common culture conditions on viability, function, and gene expression. Additionally, we compared three methods of RNA isolation and identified a protocol with high yield and purity. We observed significantly increased albumin production when cultured with insulin-transferrin-selenium and dexamethasone, and when incubated on a rocking platform. Culturing with insulin-transferrin-selenium and dexamethasone maintained gene expression closer to the levels in fresh slices. However, despite stable viability and function up to 4 days, we found significant changes in expression of key genes by day 2. Interestingly, we also observed that cirrhotic PCLS maintain viability in culture longer than slices from healthy animals. Due to the influence of matrix stiffness on fibrosis and hepatocellular function, it is important to evaluate prospective anti-fibrotic therapies in a platform that preserves tissue biomechanics. PCLS from cirrhotic animals represent a promising tool for the development of treatments for chronic liver disease.
Assuntos
Dexametasona , Cirrose Hepática , Fígado , Animais , Ratos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Cirrose Hepática/genética , Dexametasona/farmacologia , Masculino , RNA/isolamento & purificação , RNA/genética , RNA/metabolismo , Insulina/metabolismo , Insulina/farmacologia , Ratos Sprague-Dawley , Selênio/farmacologia , Técnicas de Cultura de Tecidos/métodosRESUMO
Background: The regulation of divalent metal transporter-1 (DMT1) by insulin has been previously described in Langerhans cells and significant neuroprotection was found by insulin and insulin-like growth factor 1 treatment during experimental cerebral ischemia in acute ischemic stroke patients and in a rat 6-OHDA model of Parkinson's disease, where DMT1 involvement is described. According to the regulation of DMT1, previously described as a target gene of NF-kB in the early phase of post-ischemic neurodegeneration, both in vitro and in vivo, and because insulin controls the NFkB signaling with protection from ischemic cell death in rat cardiomyocytes, we evaluated the role of insulin in relation to DMT1 expression and function during ischemic neurodegeneration. Methods: Insulin neuroprotection is evaluated in differentiated human neuroblastoma cells, SK-N-SH, and in primary mouse cortical neurons exposed to oxygen glucose deprivation (OGD) for 8 h or 3 h, respectively, with or without 300 nM insulin. The insulin neuroprotection during OGD was evaluated in both cellular models in terms of cell death, and in SK-N-SH for DMT1 protein expression and acute ferrous iron treatment, performed in acidic conditions, known to promote the maximum DMT1 uptake as a proton co-transporter; and the transactivation of 1B/DMT1 mouse promoter, already known to be responsive to NF-kB, was analyzed in primary mouse cortical neurons. Results: Insulin neuroprotection during OGD was concomitant to the down-regulation of both DMT1 protein expression and 1B/DMT1 mouse promoter transactivation. We also showed the insulin-dependent protection from cell death after acute ferrous iron treatment. In conclusion, although preliminary, this evaluation highlights the peculiar role of DMT1 as a possible pharmacological target, involved in neuroprotection by insulin during in vitro neuronal ischemia and acute ferrous iron uptake.
Assuntos
Proteínas de Transporte de Cátions , Morte Celular , Regulação para Baixo , Insulina , Neurônios , Animais , Insulina/metabolismo , Insulina/farmacologia , Humanos , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cátions/genética , Camundongos , Morte Celular/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Neuroproteção/efeitos dos fármacos , Linhagem Celular Tumoral , Fármacos Neuroprotetores/farmacologia , Ferro/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/patologia , Glucose/metabolismo , Compostos Ferrosos/farmacologiaAssuntos
Diabetes Mellitus , Desenho de Fármacos , Hipoglicemiantes , Insulina , Animais , Humanos , Diabetes Mellitus/sangue , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Insulina/administração & dosagem , Insulina/farmacologia , Insulina/uso terapêutico , Glicemia , Injeções SubcutâneasRESUMO
Current methods of storing explanted donor livers at 4 °C in University of Wisconsin (UW) solution result in loss of graft function and ultimately lead to less-than-ideal outcomes post transplantation. Our lab has previously shown that supplementing UW solution with 35-kilodalton polyethylene glycol (PEG) has membrane stabilizing effects for cold stored primary rat hepatocytes in suspension. Expanding on past studies, we here investigate if PEG has the same beneficial effects in an adherent primary rat hepatocyte cold storage model. In addition, we investigated the extent of cold-induced apoptosis through treating cold-stored hepatocytes with pan caspase inhibitor emricasan. In parallel to storage at the current cold storage standard of 4 °C, we investigated the effects of lowering the storage temperature to -4 °C, at which the storage solution remains ice-free due to the supercooling phenomenon. We show the addition of 5 % PEG to the storage medium significantly reduced the release of lactate dehydrogenase (LDH) in plated rat hepatocytes and a combinatorial treatment with emricasan maintains hepatocyte viability and morphology following recovery from cold storage. These results show that cold-stored hepatocytes undergo multiple mechanisms of cold-induced injury and that PEG and emricasan treatment in combination with supercooling may improve cell and organ preservation.
Assuntos
Apoptose , Inibidores de Caspase , Criopreservação , Hepatócitos , L-Lactato Desidrogenase , Soluções para Preservação de Órgãos , Polietilenoglicóis , Animais , Hepatócitos/efeitos dos fármacos , Polietilenoglicóis/farmacologia , Ratos , Soluções para Preservação de Órgãos/farmacologia , Criopreservação/métodos , Masculino , L-Lactato Desidrogenase/metabolismo , Apoptose/efeitos dos fármacos , Inibidores de Caspase/farmacologia , Rafinose/farmacologia , Células Cultivadas , Alopurinol/farmacologia , Crioprotetores/farmacologia , Temperatura Baixa , Glutationa/metabolismo , Glutationa/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Insulina/farmacologia , Adenosina/farmacologia , Preservação de Órgãos/métodos , Ratos Sprague-Dawley , Ácidos PentanoicosRESUMO
Pancreatic ß-cell mass is a critical determinant of insulin secretion. Severe endoplasmic reticulum (ER) stress causes ß-cell apoptosis; however, the mechanisms of progression and suppression are not yet fully understood. Here, we report that the autocrine/paracrine function of insulin reduces ER stress-induced ß-cell apoptosis. Insulin reduced the ER-stress inducer tunicamycin- and thapsigargin-induced cell viability loss due to apoptosis in INS-1 ß-cells. Moreover, the effect of insulin was greater than that of insulin-like growth factor-1 at physiologically relevant concentrations. Insulin did not attenuate the ER stress-induced increase in unfolded protein response genes. ER stress did not induce cytochrome c release from mitochondria. Mitochondrial hyperpolarization was induced by ER stress and prevented by insulin. The protonophore/mitochondrial oxidative phosphorylation uncoupler, but not the antioxidants N-acetylcysteine and α-tocopherol, exhibited potential cytoprotection during ER stress. Both procaspase-12 and cleaved caspase-12 levels increased under ER stress. The caspase-12 inhibitor Z-ATAD-FMK decreased ER stress-induced apoptosis. Caspase-12 overexpression reduced cell viability, which was diminished in the presence of insulin. Insulin decreased caspase-12 levels at the post-translational stages. These results demonstrate that insulin protects against ER stress-induced ß-cell apoptosis in this cell line. Furthermore, mitochondrial hyperpolarization and increased caspase-12 levels are involved in ER stress-induced and insulin-suppressed ß-cell apoptosis.
Assuntos
Apoptose , Caspase 12 , Estresse do Retículo Endoplasmático , Células Secretoras de Insulina , Insulina , Mitocôndrias , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Animais , Insulina/farmacologia , Insulina/metabolismo , Caspase 12/metabolismo , Caspase 12/genética , Ratos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacosRESUMO
Based on our hypothesis that myotubes exhibit a bistable response to insulin, here we present a protocol for finely measuring Akt phosphorylation in single myotubes under insulin stimulation. We describe steps to stably express a Förster resonance energy transfer (FRET)-based Akt biosensor in C2C12-derived myotubes and perform single-cell FRET imaging. This protocol highlights its potential for precision medicine in analyzing protein phosphorylation dynamics at the single-cell level. For complete details on the use and execution of this protocol, please refer to Akhtar et al.1.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Insulina , Fibras Musculares Esqueléticas , Transferência Ressonante de Energia de Fluorescência/métodos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/citologia , Insulina/metabolismo , Insulina/farmacologia , Animais , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linhagem Celular , Fosforilação , Análise de Célula Única/métodos , Técnicas Biossensoriais/métodosRESUMO
Objective: Insulin attaches insulin receptor to activate the PI3-kinase/Akt signaling to maintain glucose homeostasis and inhibit apoptosis. This study determined whether preconditioning with insulin and glucose protects the kidney against ischemia-reperfusion injury (IRI). Methods: Kidney IRI was performed in C57BL/6 mice by clamping the renal vessels for 30 min, followed by reperfusion for 24 h. A total subcutaneous 0.1 unit of insulin along with 10% glucose in drinking water was treated on the mice for 24 h before kidney IRI. The kidney function and injuries were investigated through the determination of BUN and Cr in blood plasma, as well as the apoptosis and the expression of P-AKT, BAX, and caspase-3 in the kidneys. The role of P-AKT in insulin-treated IRI kidneys was tested using an AKT inhibitor. The effects of the preconditional duration of insulin and glucose on IRI kidneys were investigated by expanding the treatment duration to 1, 3, and 6 days. Results: Preconditioning with insulin and glucose protected the kidney against IRI as manifested by a decrease in creatinine and BUN and a reduction of kidney tubular injury. The protection effect was mediated by P-AKT-BAX-caspase-3 signaling pathway resulting in suppression of apoptotic cell death. An AKT inhibitor partially reversed the protective effects of preconditional insulin. The preconditional duration for 1, 3, and 6 days had no differences in improving kidney functions and pathology. Conclusion: A short-term preconditioning with insulin and glucose protected the kidney from IRI through the activation of p-AKT and subsequent reduction of BAX-caspase-3-induced apoptosis. The short-term precondition provides a practicable strategy for protecting the kidney against predictable IRI, such as kidney transplant and major surgical operations with high risk of hypotension.
Assuntos
Caspase 3 , Glucose , Insulina , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt , Traumatismo por Reperfusão , Transdução de Sinais , Proteína X Associada a bcl-2 , Animais , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/prevenção & controle , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Camundongos , Transdução de Sinais/efeitos dos fármacos , Insulina/farmacologia , Masculino , Caspase 3/metabolismo , Glucose/metabolismo , Proteína X Associada a bcl-2/metabolismo , Rim/efeitos dos fármacos , Rim/patologia , Rim/metabolismo , Apoptose/efeitos dos fármacosRESUMO
AKT, also known as protein kinase B (PKB), serves as a crucial regulator of numerous biological functions, including cell growth, metabolism, and tumorigenesis. Increasing evidence suggests that the kinase activity of AKT is regulated via ubiquitination by various E3 ligase enzymes in response to different stimuli. However, the molecular mechanisms underlying insulin-induced AKT ubiquitination are not yet fully understood. Here, we show that activation of the insulin receptor (IR) leads to enhanced ubiquitination of AKT1 at K8 and K14 residues, facilitated by the cytosolic E3 ubiquitin ligase enzyme, TRAF6. Further investigation using AKT1 mutants with modified nucleocytoplasmic shuttling properties reveals that TRAF6-mediated AKT1 ubiquitination occurs within the nucleus in a ß-Arr2-dependent manner. The nuclear entry of TRAF6 depends on importin ß1, while ß-Arr2 regulates this process by facilitating the interaction between TRAF6 and importin ß1. Additionally, the ubiquitination of AKT1 is essential for its translocation to the activated IR on the plasma membrane, where it plays a functional role in recruiting Glut4 and facilitating glucose uptake. This study uncovers the cellular components and processes involved in insulin-induced ubiquitination and activation of AKT1, providing insights and detailed strategies for manipulating AKT1.
Assuntos
Núcleo Celular , Insulina , Proteínas Proto-Oncogênicas c-akt , Fator 6 Associado a Receptor de TNF , Ubiquitinação , beta-Arrestina 2 , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ubiquitinação/fisiologia , Ubiquitinação/efeitos dos fármacos , Insulina/metabolismo , Insulina/farmacologia , Animais , Fator 6 Associado a Receptor de TNF/metabolismo , Núcleo Celular/metabolismo , Camundongos , Humanos , beta-Arrestina 2/metabolismo , beta-Arrestina 2/genética , Células HEK293RESUMO
Ketone bodies (acetoacetate and ß-hydroxybutyrate) are oxidized in skeletal muscle mainly during fasting as an alternative source of energy to glucose. Previous studies suggest that there is a negative relationship between increased muscle ketolysis and muscle glucose metabolism in mice with obesity and/or type 2 diabetes. Therefore, we investigated the connection between increased ketone body exposure and muscle glucose metabolism by measuring the effect of a 3-h exposure to ketone bodies on glucose uptake in differentiated L6 myotubes. We showed that exposure to acetoacetate at a typical concentration (0.2 mM) resulted in increased basal glucose uptake in L6 myotubes, which was dependent on increased membrane glucose transporter type 4 (GLUT4) translocation. Basal and insulin-stimulated glucose uptake was also increased with a concentration of acetoacetate reflective of diabetic ketoacidosis or a ketogenic diet (1 mM). We found that ß-hydroxybutyrate had a variable effect on basal glucose uptake: a racemic mixture of the two ß-hydroxybutyrate enantiomers (d and l) appeared to decrease basal glucose uptake, while 3 mM d-ß-hydroxybutyrate alone increased basal glucose uptake. However, the effects of the ketone bodies individually were not observed when acetoacetate was present in combination with ß-hydroxybutyrate. These results provide insight that will help elucidate the effect of ketone bodies in the context of specific metabolic diseases and nutritional states (e.g., type 2 diabetes and ketogenic diets).NEW & NOTEWORTHY A limited number of studies investigate the effect of ketone bodies at concentrations reflective of both typical fasting and ketoacidosis. We tested a mix of physiologically relevant concentrations of ketone bodies, which allowed us to highlight the differential effects of d- and l-ß-hydroxybutyrate and acetoacetate on skeletal muscle cell glucose uptake. Our findings will assist in better understanding the mechanisms that contribute to muscle insulin resistance and provide guidance on recommendations regarding ketogenic diets.
Assuntos
Ácido 3-Hidroxibutírico , Acetoacetatos , Glucose , Insulina , Fibras Musculares Esqueléticas , Acetoacetatos/metabolismo , Acetoacetatos/farmacologia , Animais , Ácido 3-Hidroxibutírico/farmacologia , Ácido 3-Hidroxibutírico/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Insulina/farmacologia , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Linhagem Celular , Músculo Esquelético/metabolismo , Músculo Esquelético/efeitos dos fármacos , Transportador de Glucose Tipo 4/metabolismo , Ratos , Corpos Cetônicos/metabolismo , CamundongosRESUMO
The 5xFAD mouse model shows age-related weight loss as well as cognitive and motor deficits. Metabolic dysregulation, especially impaired insulin signaling, is also present in AD. This study examined whether intranasal delivery of insulin (INI) at low (0.875 U) or high (1.750 U) doses would ameliorate these deficits compared to saline in 10-month-old female 5xFAD and B6SJL wildtype (WT) mice. INI increased forelimb grip strength in the wire hang test in 5xFAD mice in a dose-dependent manner but did not improve the performance of 5xFAD mice on the balance beam. High INI doses reduced frailty scores in 5xFAD mice and improved spatial memory in both acquisition and reversal probe trials in the Morris water maze. INI increased swim speed in 5xFAD mice but had no effect on object recognition memory or working memory in the spontaneous alternation task, nor did it improve memory in the contextual or cued fear memory tasks. High doses of insulin increased the liver, spleen, and kidney weights and reduced brown adipose tissue weights. P-Akt signaling in the hippocampus was increased by insulin in a dose-dependent manner. Altogether, INI increased strength, reduced frailty scores, and improved visual spatial memory. Hypoglycemia was not present after INI, however alterations in tissue and organ weights were present. These results are novel and important as they indicate that intra-nasal insulin can reverse cognitive, motor and frailty deficits found in this mouse model of AD.
Assuntos
Administração Intranasal , Modelos Animais de Doenças , Fragilidade , Insulina , Camundongos Transgênicos , Força Muscular , Memória Espacial , Animais , Insulina/administração & dosagem , Insulina/farmacologia , Força Muscular/efeitos dos fármacos , Memória Espacial/efeitos dos fármacos , Feminino , Fragilidade/tratamento farmacológico , Camundongos , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/farmacologia , Doença de Alzheimer/tratamento farmacológico , Aprendizagem em Labirinto/efeitos dos fármacos , Relação Dose-Resposta a Droga , Transtornos da Memória/tratamento farmacológico , Precursor de Proteína beta-Amiloide/genética , Força da Mão/fisiologia , Medo/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismoRESUMO
INTRODUCTION: Anorexia Nervosa (AN) is a psycho-socio-biological disease characterized by severe weight loss as result of dieting and hyperactivity. Effective treatments are scarce, despite its significant prevalence and mortality. AN patients show lower basal insulin levels and increased metabolic clearance, leading to weight loss, cognitive deficits, and hormonal imbalances. Low-dose polymer insulin could potentially reverse these effects by restoring brain function, reducing fear of weight gain, encouraging food intake, and restoring fat depots. This study evaluates an insulin delivery system designed for sustained release and AN treatment. METHODS: AN-like model was established through dietary restriction (DR). On days 1-25, mice were on DR, and on days 26-31 they were on ad libitum regimen. An insulin-loaded delivery system was administered subcutaneously (1% w/w insulin). The impact of insulin treatment on gene expression in the hippocampus (cognition, regulation of stress, neurogenesis) and hypothalamus (eating behavior, mood) was assessed. Behavioral assays were conducted to evaluate motor activity and cognitive function. RESULTS: The delivery system demonstrated sustained insulin release, maintaining therapeutic plasma levels. Diet restriction mice treated with the insulin delivery system showed body weight restoration. Gene expression analysis revealed enhanced expression of CB1 and CB2 genes associated with improved eating behavior and cognition, while POMC expression was reduced. Insulin-polymer treatment restored cognitive function and decreased hyperactivity in the AN-like model. CONCLUSION: The PSA-RA-based insulin delivery system effectively restores metabolic balance, body weight, and cognitive function in the AN model. Its ability to steadily release insulin makes it a promising candidate for AN treatment."
Assuntos
Anorexia Nervosa , Peso Corporal , Modelos Animais de Doenças , Insulina , Animais , Insulina/administração & dosagem , Insulina/farmacologia , Camundongos , Anorexia Nervosa/tratamento farmacológico , Anorexia Nervosa/metabolismo , Peso Corporal/efeitos dos fármacos , Cognição/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/efeitos dos fármacos , Feminino , Hipotálamo/metabolismo , Hipotálamo/efeitos dos fármacos , Camundongos Endogâmicos C57BLRESUMO
Evidence suggests that insulin resistance plays an important role in developing diabetes complications. The association between insulin resistance and pain perception is less well understood. This study aimed to investigate the effects of peripheral insulin deficiency on pain pathways in the brain. Diabetes was induced in 60 male rats with streptozotocin (STZ). Insulin was injected into the left ventricle of the brain by intracerebroventricular (ICV) injection, then pain was induced by subcutaneous injection of 2.5% formalin. Samples were collected at 4 weeks after STZ injection. Dopamine (DA), serotonin, reactive oxygen species (ROS), and mitochondrial glutathione (mGSH) were measured by ELISA, and gene factors were assessed by RT-qPCR. In diabetic rats, the levels of DA, serotonin, and mGSH decreased in the nuclei of the thalamus, raphe magnus, and periaqueductal gray, and the levels of ROS increased. In addition, the levels of expression of the neuron-specific enolase and receptor for advanced glycation end genes increased, but the expression of glial fibrillary acidic protein expression was reduced. These results support the findings that insulin has an analgesic effect in non-diabetic rats, as demonstrated by the formalin test. ICV injection of insulin reduces pain sensation, but this was not observed in diabetic rats, which may be due to cell damage ameliorated by insulin.
Assuntos
Diabetes Mellitus Experimental , Resistência à Insulina , Ratos , Masculino , Animais , Insulina/farmacologia , Estreptozocina , Diabetes Mellitus Experimental/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serotonina , Dor/tratamento farmacológico , Analgésicos/efeitos adversosRESUMO
INTRODUCTION: Glucagon receptor agonism is currently explored for the treatment of obesity and metabolic dysfunction-associated steatotic liver disease (MASLD). The metabolic effects of glucagon receptor agonism may in part be mediated by increases in circulating levels of Fibroblast Growth Factor 21 (FGF21) and Growth Differentiation Factor 15 (GDF15). The effect of glucagon agonism on FGF21 and GDF15 levels remains uncertain, especially in the context of elevated insulin levels commonly observed in metabolic diseases. METHODS: We investigated the effect of a single bolus of glucagon and a continuous infusion of glucagon on plasma concentrations of FGF21 and GDF15 in conditions of endogenous low or high insulin levels. The studies included individuals with overweight with and without MASLD, healthy controls (CON) and individuals with type 1 diabetes (T1D). The direct effect of glucagon on FGF21 and GDF15 was evaluated using our in-house developed isolated perfused mouse liver model. RESULTS: FGF21 and GDF15 correlated with plasma levels of insulin, but not glucagon, and their secretion was highly increased in MASLD compared with CON and T1D. Furthermore, FGF21 levels in individuals with overweight with or without MASLD did not increase after glucagon stimulation when insulin levels were kept constant. FGF21 and GDF15 levels were unaffected by direct stimulation with glucagon in the isolated perfused mouse liver. CONCLUSION: The glucagon-induced secretion of FGF21 and GDF15 is augmented in MASLD and may depend on insulin. Thus, glucagon receptor agonism may augment its metabolic benefits in patients with MASLD through enhanced secretion of FGF21 and GDF15.