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Caspases are cysteine-aspartic proteases that were initially discovered to play a role in apoptosis. However, caspase 8, in particular, also has additional nonapoptotic roles, such as in inflammation. Adipocyte cell death and inflammation are hypothesized to be initiating pathogenic factors in type 2 diabetes. Here, we examined the pleiotropic role of caspase 8 in adipocytes and obesity-associated insulin resistance. Caspase 8 expression was increased in adipocytes from mice and humans with obesity and insulin resistance. Treatment of 3T3-L1 adipocytes with caspase 8 inhibitor Z-IETD-FMK decreased both death receptor-mediated signaling and targets of nuclear factor κ-light-chain-enhancer of activated B (NF-κB) signaling. We generated novel adipose tissue and adipocyte-specific caspase 8 knockout mice (aP2Casp8-/- and adipoqCasp8-/-). Both males and females had improved glucose tolerance in the setting of high-fat diet (HFD) feeding. Knockout mice also gained less weight on HFD, with decreased adiposity, adipocyte size, and hepatic steatosis. These mice had decreased adipose tissue inflammation and decreased activation of canonical and noncanonical NF-κB signaling. Furthermore, they demonstrated increased energy expenditure, core body temperature, and UCP1 expression. Adipocyte-specific activation of Ikbkb or housing mice at thermoneutrality attenuated improvements in glucose tolerance. These data demonstrate an important role for caspase 8 in mediating adipocyte cell death and inflammation to regulate glucose and energy homeostasis. ARTICLE HIGHLIGHTS: Caspase 8 is increased in adipocytes from mice and humans with obesity and insulin resistance. Knockdown of caspase 8 in adipocytes protects mice from glucose intolerance and weight gain on a high-fat diet. Knockdown of caspase 8 decreases Fas signaling, as well as canonical and noncanonical nuclear factor κ-light-chain-enhancer of activated B (NF-κB) signaling in adipose tissue. Improved glucose tolerance occurs via reduced activation of NF-κB signaling and via induction of UCP1 in adipocytes.
Assuntos
Diabetes Mellitus Tipo 2 , Resistência à Insulina , Humanos , Masculino , Feminino , Animais , Camundongos , NF-kappa B/metabolismo , Resistência à Insulina/genética , Caspase 8/genética , Caspase 8/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Camundongos Knockout , Adipócitos/metabolismo , Obesidade/genética , Obesidade/metabolismo , Dieta Hiperlipídica/efeitos adversos , Inflamação/metabolismo , Glucose/metabolismo , Apoptose/genéticaRESUMO
Inflammation and tissue fibrosis co-exist and are causally linked to organ dysfunction. However, the molecular mechanisms driving immune-fibroblast crosstalk in human cardiac disease remains unexplored and there are currently no therapeutics to target fibrosis. Here, we performed multi-omic single-cell gene expression, epitope mapping, and chromatin accessibility profiling in 38 donors, acutely infarcted, and chronically failing human hearts. We identified a disease-associated fibroblast trajectory marked by cell surface expression of fibroblast activator protein (FAP), which diverged into distinct myofibroblasts and pro-fibrotic fibroblast populations, the latter resembling matrifibrocytes. Pro-fibrotic fibroblasts were transcriptionally similar to cancer associated fibroblasts and expressed high levels of collagens and periostin (POSTN), thymocyte differentiation antigen 1 (THY-1), and endothelin receptor A (EDNRA) predicted to be driven by a RUNX1 gene regulatory network. We assessed the applicability of experimental systems to model tissue fibrosis and demonstrated that 3 different in vivo mouse models of cardiac injury were superior compared to cultured human heart and dermal fibroblasts in recapitulating the human disease phenotype. Ligand-receptor analysis and spatial transcriptomics predicted that interactions between C-C chemokine receptor type 2 (CCR2) macrophages and fibroblasts mediated by interleukin 1 beta (IL-1ß) signaling drove the emergence of pro-fibrotic fibroblasts within spatially defined niches. This concept was validated through in silico transcription factor perturbation and in vivo inhibition of IL-1ß signaling in fibroblasts where we observed reduced pro-fibrotic fibroblasts, preferential differentiation of fibroblasts towards myofibroblasts, and reduced cardiac fibrosis. Herein, we show a subset of macrophages signal to fibroblasts via IL-1ß and rewire their gene regulatory network and differentiation trajectory towards a pro-fibrotic fibroblast phenotype. These findings highlight the broader therapeutic potential of targeting inflammation to treat tissue fibrosis and restore organ function.
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PURPOSE OF REVIEW: Cardiac fibroblast activation contributes to fibrosis, maladaptive remodeling and heart failure progression. This review summarizes the latest findings on cardiac fibroblast activation dynamics derived from single-cell transcriptomic analyses and discusses how this information may aid the development of new multispecific medicines. RECENT FINDINGS: Advances in single-cell gene expression technologies have led to the discovery of distinct fibroblast subsets, some of which are more prevalent in diseased tissue and exhibit temporal changes in response to injury. In parallel to the rapid development of single-cell platforms, the advent of multispecific therapeutics is beginning to transform the biopharmaceutical landscape, paving the way for the selective targeting of diseased fibroblast subpopulations. Insights gained from single-cell technologies reveal critical cardiac fibroblast subsets that play a pathogenic role in the progression of heart failure. Combined with the development of multispecific therapeutic agents that have enabled access to previously "undruggable" targets, we are entering a new era of precision medicine.
Assuntos
Miocárdio , Medicina de Precisão , Fibroblastos/patologia , Fibrose , Coração , Humanos , Miocárdio/patologiaRESUMO
Inflammation is a key contributor to atherosclerosis with macrophages playing a pivotal role through the induction of oxidative stress and cytokine/chemokine secretion. DJ1, an anti-oxidant protein, has shown to paradoxically protect against chronic and acute inflammation. However, the role of DJ1 in atherosclerosis remains elusive. To assess the role of Dj1 in atherogenesis, we generated whole-body Dj1-deficient atherosclerosis-prone Apoe null mice (Dj1-/-Apoe-/-). After 21 weeks of atherogenic diet, Dj1-/- Apoe-/-mice were protected against atherosclerosis with significantly reduced plaque macrophage content. To assess whether haematopoietic or parenchymal Dj1 contributed to atheroprotection in Dj1-deficient mice, we performed bone-marrow (BM) transplantation and show that Dj1-deficient BM contributed to their attenuation in atherosclerosis. To assess cell-autonomous role of macrophage Dj1 in atheroprotection, BM-derived macrophages from Dj1-deficient mice and Dj1-silenced macrophages were assessed in response to oxidized low-density lipoprotein (oxLDL). In both cases, there was an enhanced anti-inflammatory response which may have contributed to atheroprotection in Dj1-deficient mice. There was also an increased trend of plasma DJ-1 levels from individuals with ischemic heart disease compared to those without. Our findings indicate an atheropromoting role of Dj1 and suggests that targeting Dj1 may provide a novel therapeutic avenue for atherosclerosis treatment or prevention.
Assuntos
Aterosclerose/genética , Inflamação/genética , Proteína Desglicase DJ-1/genética , Animais , Células Cultivadas , Feminino , Deleção de Genes , Humanos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Fatores de Proteção , Células RAW 264.7RESUMO
Endocrine fibroblast growth factors (FGFs) require Klotho transmembrane proteins as necessary co-receptors to activate FGF receptor (FGFR) signaling. In particular, FGF19 and FGF21 function through ß-Klotho to regulate glucose and lipid metabolism. Recent research has focused on elucidating how these two FGFs interact with ß-Klotho and FGFRs to activate downstream signaling. In this study, using hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS), we identified regions on the ß-Klotho protein that likely participate in ligand interaction, and vice versa. Alanine and arginine mutagenesis were carried out to further probe the contributions of individual residues to receptor/ligand interactions. Using biochemical and cell-based signaling assays with full-length proteins, we show that both the KL1 and KL2 domains of ß-Klotho participate in ligand interaction, and these binding sites on ß-Klotho are shared by FGF19 and FGF21. In addition, we show that two highly conserved regions in the C-terminal tail of FGF19 and FGF21 are responsible for interaction with the co-receptor. Our results are consistent with recent publications on the crystal structures of the Klotho proteins and provide insight into how endocrine FGFs interact with co-receptors for signal transduction.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais/fisiologia , Ensaio de Imunoadsorção Enzimática , Fatores de Crescimento de Fibroblastos/genética , Células HEK293 , Humanos , Proteínas Klotho , Espectrometria de Massas , Proteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genéticaRESUMO
Bispecific antibodies have become important formats for therapeutic discovery. They allow for potential synergy by simultaneously engaging two separate targets and enable new functions that are not possible to achieve by using a combination of two monospecific antibodies. Antagonistic antibodies dominate drug discovery today, but only a limited number of agonistic antibodies (i.e. those that activate receptor signaling) have been described. For receptors formed by two components, engaging both of these components simultaneously may be required for agonistic signaling. As such, bispecific antibodies may be particularly useful in activating multicomponent receptor complexes. Here, we describe a biparatopic (i.e. targeting two different epitopes on the same target) format that can activate the endocrine fibroblast growth factor (FGF) 21 receptor (FGFR) complex containing ß-Klotho and FGFR1c. This format was constructed by grafting two different antigen-specific VH domains onto the VH and VL positions of an IgG, yielding a tetravalent binder with two potential geometries, a close and a distant, between the two paratopes. Our results revealed that the biparatopic molecule provides activities that are not observed with each paratope alone. Our approach could help address the challenges with heterogeneity inherent in other bispecific formats and could provide the means to adjust intramolecular distances of the antibody domains to drive optimal activity in a bispecific format. In conclusion, this format is versatile, is easy to construct and produce, and opens a new avenue for agonistic antibody discovery and development.
Assuntos
Anticorpos Biespecíficos/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas de Membrana/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Sítios de Ligação de Anticorpos , Linhagem Celular , Epitopos/metabolismo , Humanos , Proteínas Klotho , Ligantes , Ratos , Anticorpos de Cadeia Única/metabolismoRESUMO
Atherosclerosis is considered both a metabolic and inflammatory disease; however, the specific tissue and signaling molecules that instigate and propagate this disease remain unclear. The liver is a central site of inflammation and lipid metabolism that is critical for atherosclerosis, and JAK2 is a key mediator of inflammation and, more recently, of hepatic lipid metabolism. However, precise effects of hepatic Jak2 on atherosclerosis remain unknown. We show here that hepatic Jak2 deficiency in atherosclerosis-prone mouse models exhibited accelerated atherosclerosis with increased plaque macrophages and decreased plaque smooth muscle cell content. JAK2's essential role in growth hormone signalling in liver that resulted in reduced IGF-1 with hepatic Jak2 deficiency played a causal role in exacerbating atherosclerosis. As such, restoring IGF-1 either pharmacologically or genetically attenuated atherosclerotic burden. Together, our data show hepatic Jak2 to play a protective role in atherogenesis through actions mediated by circulating IGF-1 and, to our knowledge, provide a novel liver-centric mechanism in atheroprotection.
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Focal adhesion kinase (FAK) plays a central role in integrin signalling, which regulates growth and survival of tumours. Here we show that FAK protein levels are increased in adipose tissue of insulin-resistant obese mice and humans. Disruption of adipocyte FAK in mice or in 3T3 L1 cells decreases adipocyte survival. Adipocyte-specific FAK knockout mice display impaired adipose tissue expansion and insulin resistance on prolonged metabolic stress from a high-fat diet or when crossed on an obese db/db or ob/ob genetic background. Treatment of these mice with a PPARγ agonist does not restore adiposity or improve insulin sensitivity. In contrast, inhibition of apoptosis, either genetically or pharmacologically, attenuates adipocyte death, restores normal adiposity and improves insulin sensitivity. Together, these results demonstrate that FAK is required for adipocyte survival and maintenance of insulin sensitivity, particularly in the context of adipose tissue expansion as a result of caloric excess.
Assuntos
Adipócitos/fisiologia , Quinase 1 de Adesão Focal/metabolismo , Resistência à Insulina/fisiologia , Obesidade/metabolismo , Células 3T3-L1 , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Tecido Adiposo/fisiopatologia , Adiposidade/efeitos dos fármacos , Adiposidade/genética , Adulto , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Feminino , Quinase 1 de Adesão Focal/genética , Humanos , Hipoglicemiantes/farmacologia , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Pessoa de Meia-Idade , Obesidade/etiologia , Obesidade/fisiopatologia , PPAR gama/agonistas , Cultura Primária de Células , Rosiglitazona , Transdução de Sinais/fisiologia , Tiazolidinedionas/farmacologiaRESUMO
Hepatocellular carcinoma is an end-stage complication of non-alcoholic fatty liver disease (NAFLD). Inflammation plays a critical role in the progression of non-alcoholic fatty liver disease and the development of hepatocellular carcinoma. However, whether steatosis per se promotes liver cancer, and the molecular mechanisms that control the progression in this disease spectrum remain largely elusive. The Janus kinase signal transducers and activators of transcription (JAK-STAT) pathway mediates signal transduction by numerous cytokines that regulate inflammation and may contribute to hepatocarcinogenesis. Mice with hepatocyte-specific deletion of JAK2 (L-JAK2 KO) develop extensive fatty liver spontaneously. We show here that this simple steatosis was insufficient to drive carcinogenesis. In fact, L-JAK2 KO mice were markedly protected from chemically induced tumor formation. Using the methionine choline-deficient dietary model to induce steatohepatitis, we found that steatohepatitis development was completely arrested in L-JAK2 KO mice despite the presence of steatosis, suggesting that JAK2 is the critical factor required for inflammatory progression in the liver. In line with this, L-JAK2 KO mice exhibited attenuated inflammation after chemical carcinogen challenge. This was associated with increased hepatocyte apoptosis without elevated compensatory proliferation, thus thwarting expansion of transformed hepatocytes. Taken together, our findings identify an indispensable role of JAK2 in hepatocarcinogenesis through regulating critical inflammatory pathways. Targeting the JAK-STAT pathway may provide a novel therapeutic option for the treatment of hepatocellular carcinoma.
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Carcinoma Hepatocelular/metabolismo , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Neoplasias Hepáticas/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Animais , Proliferação de Células , Fígado Gorduroso/metabolismo , Deleção de Genes , Hepatócitos/metabolismo , Inflamação , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Obesity-related inflammation of metabolic tissues, including visceral adipose tissue (VAT) and liver, are key factors in the development of insulin resistance (IR), though many of the contributing mechanisms remain unclear. We show that nucleic-acid-targeting pathways downstream of extracellular trap (ET) formation, unmethylated CpG DNA, or ribonucleic acids drive inflammation in IR. High-fat diet (HFD)-fed mice show increased release of ETs in VAT, decreased systemic clearance of ETs, and increased autoantibodies against conserved nuclear antigens. In HFD-fed mice, this excess of nucleic acids and related protein antigens worsens metabolic parameters through a number of mechanisms, including activation of VAT macrophages and expansion of plasmacytoid dendritic cells (pDCs) in the liver. Consistently, HFD-fed mice lacking critical responders of nucleic acid pathways, Toll-like receptors (TLR)7 and TLR9, show reduced metabolic inflammation and improved glucose homeostasis. Treatment of HFD-fed mice with inhibitors of ET formation or a TLR7/9 antagonist improves metabolic disease. These findings reveal a pathogenic role for nucleic acid targeting as a driver of metabolic inflammation in IR.
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Inflamação/metabolismo , Inflamação/patologia , Resistência à Insulina/fisiologia , Ácidos Nucleicos/metabolismo , Obesidade/metabolismo , Obesidade/patologia , Adulto , Animais , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Dieta Hiperlipídica/métodos , Glucose/metabolismo , Homeostase/fisiologia , Humanos , Gordura Intra-Abdominal/metabolismo , Gordura Intra-Abdominal/patologia , Fígado/metabolismo , Fígado/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Receptores Toll-Like/metabolismoRESUMO
AIMS/HYPOTHESIS: Non-shivering thermogenesis in adipose tissue can be activated by excessive energy intake or following cold exposure. The molecular mechanisms regulating this activation have not been fully elucidated. The Janus kinase (JAK) - signal transducer and activator of transcription (STAT) pathway mediates the signal transduction of numerous hormones and growth factors that regulate adipose tissue development and function, and may play a role in adaptive thermogenesis. METHODS: We analysed mRNA and protein levels of uncoupling protein 1 (UCP1) and JAK2 in different adipose depots in response to metabolic and thermal stress. The in vivo role of JAK2 in adaptive thermogenesis was examined using mice with adipocyte-specific Jak2 deficiency (A-Jak2 KO). RESULTS: We show in murine brown adipose tissue (BAT) that JAK2 is upregulated together with UCP1 in response to high-fat diet (HFD) feeding and cold exposure. In contrast to white adipose tissue, where JAK2 was dispensable for UCP1 induction, we identified an essential role for BAT JAK2 in diet- and cold-induced thermogenesis via mediating the thermogenic response to ß-adrenergic stimulation. Accordingly, A-Jak2 KO mice were unable to upregulate BAT UCP1 following a HFD or after cold exposure. Therefore, A-Jak2 KO mice were cold intolerant and susceptible to HFD-induced obesity and diabetes. CONCLUSIONS/INTERPRETATION: Taken together, our results suggest that JAK2 plays a critical role in BAT function and adaptive thermogenesis. Targeting the JAK-STAT pathway may be a novel therapeutic approach for the treatment of obesity and related metabolic disorders.
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Tecido Adiposo Marrom/fisiologia , Janus Quinase 2/metabolismo , Termogênese , Adipócitos/citologia , Adipogenia , Tecido Adiposo Branco/fisiologia , Adiposidade , Animais , Dieta Hiperlipídica , Feminino , Insulina/fisiologia , Canais Iônicos/fisiologia , Janus Quinase 1/fisiologia , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/fisiologia , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Fator de Transcrição STAT1/fisiologia , Transdução de Sinais , Proteína Desacopladora 1 , Regulação para CimaRESUMO
Reactive oxygen species (ROS) have been linked to a wide variety of pathologies, including obesity and diabetes, but ROS also act as endogenous signalling molecules, regulating numerous biological processes. DJ-1 is one of the most evolutionarily conserved proteins across species, and mutations in DJ-1 have been linked to some cases of Parkinson's disease. Here we show that DJ-1 maintains cellular metabolic homeostasis via modulating ROS levels in murine skeletal muscles, revealing a role of DJ-1 in maintaining efficient fuel utilization. We demonstrate that, in the absence of DJ-1, ROS uncouple mitochondrial respiration and activate AMP-activated protein kinase, which triggers Warburg-like metabolic reprogramming in muscle cells. Accordingly, DJ-1 knockout mice exhibit higher energy expenditure and are protected from obesity, insulin resistance and diabetes in the setting of fuel surplus. Our data suggest that promoting mitochondrial uncoupling may be a potential strategy for the treatment of obesity-associated metabolic disorders.
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Metabolismo Energético/genética , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Mioblastos Esqueléticos/metabolismo , Proteínas Oncogênicas/genética , Peroxirredoxinas/genética , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Linhagem Celular , Sobrevivência Celular , Diabetes Mellitus/genética , Dieta Hiperlipídica , Glucose/metabolismo , Glicólise/genética , Homeostase/genética , Immunoblotting , Resistência à Insulina/genética , Camundongos , Camundongos Knockout , Fibras Musculares Esqueléticas/metabolismo , Obesidade/genética , Estresse Oxidativo , Consumo de Oxigênio , Proteína Desglicase DJ-1RESUMO
Obesity has reached epidemic proportions, but little is known about its influence on the intestinal immune system. Here we show that the gut immune system is altered during high-fat diet (HFD) feeding and is a functional regulator of obesity-related insulin resistance (IR) that can be exploited therapeutically. Obesity induces a chronic phenotypic pro-inflammatory shift in bowel lamina propria immune cell populations. Reduction of the gut immune system, using beta7 integrin-deficient mice (Beta7(null)), decreases HFD-induced IR. Treatment of wild-type HFD C57BL/6 mice with the local gut anti-inflammatory, 5-aminosalicyclic acid (5-ASA), reverses bowel inflammation and improves metabolic parameters. These beneficial effects are dependent on adaptive and gut immunity and are associated with reduced gut permeability and endotoxemia, decreased visceral adipose tissue inflammation, and improved antigen-specific tolerance to luminal antigens. Thus, the mucosal immune system affects multiple pathways associated with systemic IR and represents a novel therapeutic target in this disease.
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Anti-Inflamatórios/farmacologia , Trato Gastrointestinal/imunologia , Imunidade nas Mucosas/imunologia , Resistência à Insulina/imunologia , Obesidade/imunologia , Animais , Western Blotting , Citocinas/sangue , Dieta Hiperlipídica/efeitos adversos , Citometria de Fluxo , Trato Gastrointestinal/efeitos dos fármacos , Técnicas Histológicas , Imuno-Histoquímica , Cadeias beta de Integrinas/metabolismo , Mesalamina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Mucosa/citologia , Mucosa/imunologiaRESUMO
Obesity-related insulin resistance is associated with an influx of pathogenic T cells into visceral adipose tissue (VAT), but the mechanisms regulating lymphocyte balance in such tissues are unknown. Here we describe an important role for the immune cytotoxic effector molecule perforin in regulating this process. Perforin-deficient mice (Prf1(null)) show early increased body weight and adiposity, glucose intolerance, and insulin resistance when placed on high-fat diet (HFD). Regulatory effects of perforin on glucose tolerance are mechanistically linked to the control of T-cell proliferation and cytokine production in inflamed VAT. HFD-fed Prf1(null) mice have increased accumulation of proinflammatory IFN-γ-producing CD4(+) and CD8(+) T cells and M1-polarized macrophages in VAT. CD8(+) T cells from the VAT of Prf1(null) mice have increased proliferation and impaired early apoptosis, suggesting a role for perforin in the regulation of T-cell turnover during HFD feeding. Transfer of CD8(+) T cells from Prf1(null) mice into CD8-deficient mice (CD8(null)) resulted in worsening of metabolic parameters compared with wild-type donors. Improved metabolic parameters in HFD natural killer (NK) cell-deficient mice (NK(null)) ruled out a role for NK cells as a single source of perforin in regulating glucose homeostasis. The findings support the importance of T-cell function in insulin resistance and suggest that modulation of lymphocyte homeostasis in inflamed VAT is one possible avenue for therapeutic intervention.
Assuntos
Intolerância à Glucose/imunologia , Resistência à Insulina/imunologia , Gordura Intra-Abdominal/imunologia , Obesidade/imunologia , Paniculite/imunologia , Perforina/imunologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Dieta Hiperlipídica , Células-Tronco Embrionárias/citologia , Feminino , Intolerância à Glucose/genética , Intolerância à Glucose/metabolismo , Insulina/metabolismo , Gordura Intra-Abdominal/metabolismo , Linfonodos/citologia , Linfonodos/imunologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/genética , Obesidade/metabolismo , Paniculite/genética , Paniculite/metabolismo , Perforina/genética , Perforina/metabolismo , Baço/citologia , Baço/imunologiaRESUMO
An aberrant increase in circulating catabolic hormone glucagon contributes to type 2 diabetes pathogenesis. However, mechanisms regulating glucagon secretion and α-cell mass are not well understood. In this study, we aimed to demonstrate that phosphatidylinositol 3-kinase (PI3K) signaling is an important regulator of α-cell function. Mice with deletion of PTEN, a negative regulator of this pathway, in α-cells show reduced circulating glucagon levels and attenuated l-arginine-stimulated glucagon secretion both in vivo and in vitro. This hypoglucagonemic state is maintained after high-fat-diet feeding, leading to reduced expression of hepatic glycogenolytic and gluconeogenic genes. These beneficial effects protected high-fat diet-fed mice against hyperglycemia and insulin resistance. The data demonstrate an inhibitory role of PI3K signaling on α-cell function and provide experimental evidence for enhancing α-cell PI3K signaling for diabetes treatment.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Glucagon/fisiologia , Glucagon/sangue , Resistência à Insulina/fisiologia , PTEN Fosfo-Hidrolase/genética , Animais , Arginina/metabolismo , Diabetes Mellitus Tipo 2/genética , Dieta Hiperlipídica , Feminino , Glucagon/metabolismo , Células Secretoras de Glucagon/citologia , Células Secretoras de Glucagon/metabolismo , Hiperglicemia/genética , Hiperglicemia/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/fisiologia , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais/fisiologiaRESUMO
AIMS/HYPOTHESIS: Diabetes mellitus is characterised by beta cell loss and alpha cell expansion. Analogues of glucagon-like peptide-1 (GLP-1) are used therapeutically to antagonise these processes; thus, we hypothesised that the related cell cycle regulators retinoblastoma protein (Rb) and p107 were involved in GLP-1 action. METHODS: We used small interfering RNA and adenoviruses to manipulate Rb and p107 expression in insulinoma and alpha-TC cell lines. In vivo we examined pancreas-specific Rb knockout, whole-body p107 knockout and Rb/p107 double-knockout mice. RESULTS: Rb, but not p107, was downregulated in response to the GLP-1 analogue, exendin-4, in both alpha and beta cells. Intriguingly, this resulted in opposite outcomes of cell cycle arrest in alpha cells but proliferation in beta cells. Overexpression of Rb in alpha and beta cells abolished or attenuated the effects of exendin-4 supporting the important role of Rb in GLP-1 modulation of cell cycling. Similarly, in vivo, Rb, but not p107, deficiency was required for the beta cell proliferative response to exendin-4. Consistent with this finding, Rb, but not p107, was suppressed in islets from humans with diabetes, suggesting the importance of Rb regulation for the compensatory proliferation that occurs under insulin resistant conditions. Finally, while p107 alone did not have an essential role in islet homeostasis, when combined with Rb deletion, its absence potentiated apoptosis of both alpha and beta cells resulting in glucose intolerance and diminished islet mass with ageing. CONCLUSIONS/INTERPRETATION: We found a central role of Rb in the dual effects of GLP-1 in alpha and beta cells. Our findings highlight unique contributions of individual Rb family members to islet cell proliferation and survival.
Assuntos
Ciclo Celular/fisiologia , Sobrevivência Celular/fisiologia , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Proteína do Retinoblastoma/metabolismo , Proteína p107 Retinoblastoma-Like/metabolismo , Animais , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Exenatida , Células Secretoras de Glucagon/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Homeostase/fisiologia , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Camundongos Knockout , Peptídeos/farmacologia , Proteína do Retinoblastoma/genética , Proteína p107 Retinoblastoma-Like/genética , Peçonhas/farmacologiaRESUMO
AIMS/HYPOTHESIS: The growing obesity epidemic necessitates a better understanding of adipocyte biology and its role in metabolism. The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway mediates signalling by numerous cytokines and hormones that regulate adipocyte function, illustrating the physiological importance of adipose JAK-STAT. The aim of this study was to investigate potential roles of adipocyte JAK2, an essential player in the JAK-STAT pathway, in adipocyte biology and metabolism. METHODS: We generated adipocyte-specific Jak2 knockout (A-Jak2 KO) mice using the Cre-loxP system with Cre expression driven by the Ap2 (also known as Fabp4) promoter. RESULTS: Starting at 2-3 months of age, male and female A-Jak2 KO mice gradually gained more body weight than control littermates primarily due to increased adiposity. This was associated with reduced energy expenditure in A-Jak2 KO mice. In perigonadal adipose tissue, the expression of numerous genes involved in lipid metabolism was differentially regulated. In addition, adipose tissue from A-Jak2 KO mice displayed impaired lipolysis in response to isoprenaline, growth hormone and leptin stimulation, suggesting that adipose JAK2 directly modulates the lipolytic program. Impaired lipid homeostasis was also associated with disrupted adipokine secretion. Accordingly, while glucose metabolism was normal at 2 months of age, by 5-6 months of age, A-Jak2 KO mice had whole-body insulin resistance. CONCLUSIONS/INTERPRETATION: Our results suggest that adipocyte JAK2 plays a critical role in the regulation of adipocyte biology and whole-body metabolism. Targeting of the JAK-STAT pathway could be a novel therapeutic option for the treatment of obesity and type 2 diabetes.
Assuntos
Adipócitos/metabolismo , Envelhecimento , Resistência à Insulina , Janus Quinase 2/metabolismo , Lipólise , Adipócitos/citologia , Adipocinas , Adiposidade , Animais , Composição Corporal , Peso Corporal , Citocinas/metabolismo , Feminino , Teste de Tolerância a Glucose , Masculino , Camundongos , Camundongos Knockout , Obesidade , Regiões Promotoras GenéticasRESUMO
Both type 1 and type 2 diabetes are associated with insufficient functional ß-cell mass. Understanding intracellular signaling pathways associated with this decline is important in broadening our understanding of the disease and potential therapeutic strategies. The hypoxia inducible factor pathway (HIF) plays a critical role in cellular adaptation to hypoxic conditions. Activation of this pathway increases expression of numerous genes involved in multiple cellular processes and has been shown to impact the regulation of ß-cell function. Previously, deletion of HIF-1α or HIF-1ß in pancreatic ß-cells, as well as constitutive activation of the HIF pathway in ß-cells, was shown to result in glucose intolerance and impaired insulin secretion. The objective of this study was to delineate roles of HIF-2α overexpression in pancreatic ß-cells in vivo. We overexpressed HIF-2α in pancreatic ß-cells by employing the Cre-loxP system driven by the Pdx1 promoter to delete a stop codon. Our study revealed that pancreatic HIF-2α overexpression does not result in significant differences in glucose tolerance, insulin sensitivity or ß-cell area compared to wild-type littermates under basal conditions or after high fat diet. Together, our study shows excess HIF-2α in the pancreatic ß-cells does not play a significant role in ß-cell function and glucose homeostasis.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Glicemia/análise , Regulação da Expressão Gênica , Teste de Tolerância a Glucose , Homeostase/fisiologia , Marcação In Situ das Extremidades Cortadas , Células Secretoras de Insulina/fisiologia , Masculino , CamundongosRESUMO
The growing prevalence of obesity and diabetes necessitate a better understanding of the role of adipocyte biology in metabolism. Increasingly, erythropoietin (EPO) has been shown to have extraerythropoietic and cytoprotective roles. Exogenous administration has recently been shown to have beneficial effects on obesity and diabetes in mouse models and EPO can modulate adipogenesis and insulin signaling in 3T3-L1 adipocytes. However, its physiological role in adipocytes has not been identified. Using male and female mice with adipose tissue-specific knockdown of the EPO receptor, we determine that adipocyte EPO signaling is not essential for the maintenance of energy homeostasis or glucose metabolism. Adipose tissue-specific disruption of EPO receptor did not alter adipose tissue expansion, adipocyte morphology, insulin resistance, inflammation, or angiogenesis in vivo. In contrast to the pharmacological effects of EPO, we demonstrate that EPO signaling at physiological levels is not essential for adipose tissue regulation of metabolism.
Assuntos
Tecido Adiposo Branco/metabolismo , Metabolismo Energético , Glucose/metabolismo , Receptores da Eritropoetina/metabolismo , Tecido Adiposo Marrom/irrigação sanguínea , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/imunologia , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/irrigação sanguínea , Tecido Adiposo Branco/citologia , Tecido Adiposo Branco/patologia , Adiposidade , Adulto , Animais , Células Cultivadas , Diabetes Mellitus Tipo 2/imunologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Dieta Hiperlipídica/efeitos adversos , Feminino , Regulação da Expressão Gênica , Humanos , Resistência à Insulina , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Neovascularização Fisiológica , Obesidade/etiologia , Obesidade/imunologia , Obesidade/metabolismo , Obesidade/patologia , Receptores da Eritropoetina/genética , Organismos Livres de Patógenos EspecíficosRESUMO
Focal adhesion kinase (FAK) acts as an adaptor at the focal contacts serving as a junction between the extracellular matrix and actin cytoskeleton. Actin dynamics is known as a determinant step in insulin secretion. Additionally, FAK has been shown to regulate insulin signaling. To investigate the essential physiological role of FAK in pancreatic ß-cells in vivo, we generated a transgenic mouse model using rat insulin promoter (RIP)-driven Cre-loxP recombination system to specifically delete FAK in pancreatic ß-cells. These RIPcre(+)fak(fl/fl) mice exhibited glucose intolerance without changes in insulin sensitivity. Reduced ß-cell viability and proliferation resulting in decreased ß-cell mass was observed in these mice, which was associated with attenuated insulin/Akt (also known as protein kinase B) and extracellular signal-related kinase 1/2 signaling and increased caspase 3 activation. FAK-deficient ß-cells exhibited impaired insulin secretion with normal glucose sensing and preserved Ca(2+) influx in response to glucose, but a reduced number of docked insulin granules and insulin exocytosis were found, which was associated with a decrease in focal proteins, paxillin and talin, and an impairment in actin depolymerization. This study is the first to show in vivo that FAK is critical for pancreatic ß-cell viability and function through regulation in insulin signaling, actin dynamics, and granule trafficking.