RESUMO
The recently elucidated proresolving conjugates in tissue regeneration (CTR) maresin-CTR (MCTR), protectin-CTR (PCTR), and resolvin-CTR (RCTR), termed cysteinyl-specialized proresolving mediators (cys-SPMs) each promotes regeneration, controls infection, and accelerates resolution of inflammation. Here, we sought evidence for cys-SPM activation of primordial pathways in planaria (Dugesia japonica) regeneration that might link resolution of inflammation and regeneration. On surgical resection, planaria regeneration was enhanced with MCTR3, PCTR3, or RCTR3 (10 nM), each used for RNA sequencing. The three cys-SPMs shared up-regulation of 175 known transcripts with fold-change > 1.25 and combined false discovery rate (FDR) < 0.002, and 199 canonical pathways (FDR < 0.25), including NF-κB pathways and an ortholog of human TRAF3 (TNFR-associated factor 3). Three separate pathway analyses converged on TRAF3 up-regulation by cys-SPMs. With human macrophages, three cys-SPMs each dose-dependently increased TRAF3 expression in a cAMP-PKA-dependent manner. TRAF3 overexpression in macrophages enhanced Interleukin-10 (IL-10) and phagocytosis of Escherichia coli IL-10 also increased phagocytosis in a dose-dependent manner. Silencing of mouse TRAF3 in vivo significantly reduced IL-10 and macrophage phagocytosis. TRAF3 silencing in vivo also relieved cys-SPMs' actions in limiting polymorphonuclear neutrophil in E. coli exudates. These results identify cys-SPM-regulated pathways in planaria regeneration, uncovering a role for TRAF3/IL-10 in regulating mammalian phagocyte functions in resolution. Cys-SPM activation of TRAF3 signaling is a molecular component of both regeneration and resolution of infectious inflammation.
Assuntos
Infecções por Escherichia coli/imunologia , Escherichia coli/imunologia , Planárias/imunologia , Regeneração/imunologia , Transdução de Sinais/imunologia , Fator 3 Associado a Receptor de TNF/imunologia , Animais , Infecções por Escherichia coli/genética , Humanos , Inflamação/genética , Inflamação/imunologia , Neutrófilos/imunologia , Fagocitose , Planárias/genética , Regeneração/genética , Transdução de Sinais/genética , Fator 3 Associado a Receptor de TNF/genéticaRESUMO
Insulin and insulin-like growth factor 1 (IGF-1) receptors share many downstream signaling pathways but have unique biological effects. To define the molecular signals contributing to these distinct activities, we performed global phosphoproteomics on cells expressing either insulin receptor (IR), IGF-1 receptor (IGF1R), or chimeric IR-IGF1R receptors. We show that IR preferentially stimulates phosphorylations associated with mammalian target of rapamycin complex 1 (mTORC1) and Akt pathways, whereas IGF1R preferentially stimulates phosphorylations on proteins associated with the Ras homolog family of guanosine triphosphate hydrolases (Rho GTPases), and cell cycle progression. There were also major differences in the phosphoproteome between cells expressing IR versus IGF1R in the unstimulated state, including phosphorylation of proteins involved in membrane trafficking, chromatin remodeling, and cell cycle. In cells expressing chimeric IR-IGF1R receptors, these differences in signaling could be mapped to contributions of both the extra- and intracellular domains of these receptors. Thus, despite their high homology, IR and IGF1R preferentially regulate distinct networks of phosphorylation in both the basal and stimulated states, allowing for the unique effects of these hormones on organismal function.
Assuntos
Antígenos CD/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Adipócitos/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Feminino , Humanos , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/fisiologia , Camundongos , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Recent studies suggest that, even within a single adipose depot, there may be distinct subpopulations of adipocytes. To investigate this cellular heterogeneity, we have developed multiple conditionally immortalized clonal preadipocyte lines from white adipose tissue of mice. Analysis of these clones reveals at least three white adipocyte subpopulations. These subpopulations have differences in metabolism and differentially respond to inflammatory cytokines, insulin, and growth hormones. These also have distinct gene expression profiles and can be tracked by differential expression of three marker genes: Wilms' tumor 1, transgelin, and myxovirus 1. Lineage tracing analysis with dual-fluorescent reporter mice indicates that these adipocyte subpopulations have differences in gene expression and metabolism that mirror those observed in the clonal cell lines. Furthermore, preadipocytes and adipocytes from these subpopulations differ in their abundance in different fat depots. Thus, white adipose tissue, even in a single depot, is comprised of distinct subpopulations of white adipocytes with different physiological phenotypes. These differences in adipocyte composition may contribute to the differences in metabolic behavior and physiology of different fat depots.
Assuntos
Adipócitos Brancos/classificação , Adipócitos Brancos/citologia , Adipogenia , Tecido Adiposo/citologia , Biomarcadores/análise , Adipócitos Brancos/fisiologia , Tecido Adiposo/fisiologia , Animais , Citocinas/metabolismo , Metabolismo Energético , Hormônio do Crescimento Humano/metabolismo , Mediadores da Inflamação/metabolismo , Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Proteínas Repressoras/metabolismo , Transcriptoma , Proteínas WT1RESUMO
Opsin3 (Opn3) is a transmembrane heptahelical G protein-coupled receptor (GPCR) with the potential to produce a nonvisual photoreceptive effect. Interestingly, anatomical profiling of GPCRs reveals that Opn3 mRNA is highly expressed in adipose tissue. The photosensitive functions of Opn3 in mammals are poorly understood, and whether Opn3 has a role in fat is entirely unknown. In this study, we found that Opn3-knockout (Opn3-KO) mice were prone to diet-induced obesity and insulin resistance. At the cellular level, Opn3-KO brown adipocytes cultured in darkness had decreased glucose uptake and lower nutrient-induced mitochondrial respiration than wild-type (WT) cells. Light exposure promoted mitochondrial activity and glucose uptake in WT adipocytes but not in Opn3-KO cells. Brown adipocytes carrying a defective mutation in Opn3's putative G protein-binding domain also exhibited a reduction in glucose uptake and mitochondrial respiration in darkness. Using RNA-sequencing, we identified several novel light-sensitive and Opn3-dependent molecular signatures in brown adipocytes. Importantly, direct exposure of brown adipose tissue (BAT) to light in living mice significantly enhanced thermogenic capacity of BAT, and this effect was diminished in Opn3-KO animals. These results uncover a previously unrecognized cell-autonomous, light-sensing mechanism in brown adipocytes via Opn3-GPCR signaling that can regulate fuel metabolism and mitochondrial respiration. Our work also provides a molecular basis for developing light-based treatments for obesity and its related metabolic disorders.
Assuntos
Adipócitos Marrons/metabolismo , Metabolismo Energético , Opsinas de Bastonetes/metabolismo , Tecido Adiposo Marrom/inervação , Animais , Dieta Hiperlipídica/efeitos adversos , Regulação da Expressão Gênica , Glucose/metabolismo , Resistência à Insulina , Luz , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Mutação , Obesidade/genética , Obesidade/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Opsinas de Bastonetes/genética , Transdução de Sinais , TermogêneseRESUMO
AIM: To determine whether continuous glucose monitoring (CGM) can reduce hypoglycaemia in patients with post-bariatric hypoglycaemia (PBH). MATERIALS AND METHODS: In an open-label, nonrandomized, pre-post design with sequential assignment, CGM data were collected in 22 individuals with PBH in two sequential phases: (i) masked (no access to sensor glucose or alarms); and (ii) unmasked (access to sensor glucose and alarms for low or rapidly declining sensor glucose). Twelve participants wore the Dexcom G4 device for a total of 28 days, while 10 wore the Dexcom G6 device for a total of 20 days. RESULTS: Participants with PBH spent a lower percentage of time in hypoglycaemia over 24 hours with unmasked versus masked CGM (<3.3 mM/L, or <60 mg/dL: median [median absolute deviation {MAD}] 0.7 [0.8]% vs. 1.4 [1.7]%, P = 0.03; <3.9 mM/L, or <70 mg/dL: median [MAD] 2.9 [2.5]% vs. 4.7 [4.8]%; P = 0.04), with similar trends overnight. Sensor glucose data from the unmasked phase showed a greater percentage of time spent between 3.9 and 10 mM/L (70-180 mg/dL) (median [MAD] 94.8 [3.9]% vs. 90.8 [5.2]%; P = 0.004) and lower glycaemic variability over 24 hours (median [MAD] mean amplitude of glycaemic excursion 4.1 [0.98] vs. 4.4 [0.99] mM/L; P = 0.04). During the day, participants also spent a greater percentage of time in normoglycaemia with unmasked CGM (median [MAD] 94.2 [4.8]% vs. 90.9 [6.2]%; P = 0.005), largely due to a reduction in hyperglycaemia (>10 mM/L, or 180 mg/dL: median [MAD] 1.9 [2.2]% vs. 3.9 [3.6]%; P = 0.02). CONCLUSIONS: Real-time CGM data and alarms are associated with reductions in low sensor glucose, elevated sensor glucose, and glycaemic variability. This suggests CGM allows patients to detect hyperglycaemic peaks and imminent hypoglycaemia, allowing dietary modification and self-treatment to reduce hypoglycaemia. The use of CGM devices may improve safety in PBH, particularly for patients with hypoglycaemia unawareness.
Assuntos
Bariatria , Diabetes Mellitus Tipo 1 , Hipoglicemia , Humanos , Glicemia , Automonitorização da Glicemia , Hipoglicemia/diagnóstico , Hipoglicemia/etiologia , Hipoglicemia/prevenção & controleRESUMO
Adipose tissue is a major site of energy storage and has a role in the regulation of metabolism through the release of adipokines. Here we show that mice with an adipose-tissue-specific knockout of the microRNA (miRNA)-processing enzyme Dicer (ADicerKO), as well as humans with lipodystrophy, exhibit a substantial decrease in levels of circulating exosomal miRNAs. Transplantation of both white and brown adipose tissue-brown especially-into ADicerKO mice restores the level of numerous circulating miRNAs that are associated with an improvement in glucose tolerance and a reduction in hepatic Fgf21 mRNA and circulating FGF21. This gene regulation can be mimicked by the administration of normal, but not ADicerKO, serum exosomes. Expression of a human-specific miRNA in the brown adipose tissue of one mouse in vivo can also regulate its 3' UTR reporter in the liver of another mouse through serum exosomal transfer. Thus, adipose tissue constitutes an important source of circulating exosomal miRNAs, which can regulate gene expression in distant tissues and thereby serve as a previously undescribed form of adipokine.
Assuntos
Tecido Adiposo/metabolismo , Regulação da Expressão Gênica , MicroRNAs/sangue , MicroRNAs/metabolismo , Comunicação Parácrina , Regiões 3' não Traduzidas/genética , Adipocinas/metabolismo , Tecido Adiposo/transplante , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Marrom/transplante , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/transplante , Animais , Exossomos/genética , Fatores de Crescimento de Fibroblastos/sangue , Fatores de Crescimento de Fibroblastos/genética , Genes Reporter/genética , Teste de Tolerância a Glucose , Fígado/metabolismo , Masculino , Camundongos , MicroRNAs/genética , Modelos Biológicos , Especificidade de Órgãos/genética , RNA Mensageiro/genética , Ribonuclease III/deficiência , Ribonuclease III/genética , Transcrição GênicaAssuntos
Hipoglicemia , Antagonistas da Insulina , Insulina , Insulinoma , Neoplasias Pancreáticas , Humanos , Anticorpos/imunologia , Hipoglicemia/etiologia , Hipoglicemia/terapia , Insulinoma/complicações , Insulinoma/imunologia , Insulinoma/terapia , Neoplasias Pancreáticas/complicações , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/terapia , Insulina/imunologia , Antagonistas da Insulina/imunologia , Antagonistas da Insulina/uso terapêuticoRESUMO
Flow-limiting atherosclerotic lesions of arteries supplying the limbs are a cause of symptoms in patients with peripheral artery disease (PAD). Musculoskeletal metabolic factors also contribute to the pathophysiology of claudication, which is manifest as leg discomfort that impairs walking capacity. Accordingly, we conducted a case-control study to determine whether skeletal muscle metabolic gene expression is altered in PAD. Calf skeletal muscle gene expression of patients with PAD and healthy subjects was analyzed using microarrays. The top-ranking gene differentially expressed between PAD and controls (FDR < 0.001) was PLA2G16, which encodes adipose-specific phospholipase A2 (AdPLA) and is implicated in the maintenance of insulin sensitivity and regulation of lipid metabolism. Differential expression was confirmed by qRT-PCR; PLA2G16 was downregulated by 68% in patients with PAD (p < 0.001). Expression of Pla2g16 was then measured in control (db/+) and diabetic (db/db) mice that underwent unilateral femoral artery ligation. There was significantly reduced expression of Pla2g16 in the ischemic leg of both control and diabetic mice (by 51%), with significantly greater magnitude of reduction in the diabetic mice (by 79%). We conclude that AdPLA is downregulated in humans with PAD and in mice with hindlimb ischemia. Reduced AdPLA may contribute to impaired walking capacity in patients with PAD via its effects on skeletal muscle metabolism. Further studies are needed to fully characterize the role of AdPLA in PAD and to investigate its potential as a therapeutic target for alleviating symptoms of claudication.
Assuntos
Claudicação Intermitente/enzimologia , Isquemia/enzimologia , Músculo Esquelético/enzimologia , Doença Arterial Periférica/enzimologia , Fosfolipases A2 Independentes de Cálcio/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Idoso , Animais , Estudos de Casos e Controles , Modelos Animais de Doenças , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Resistência à Insulina , Claudicação Intermitente/genética , Claudicação Intermitente/fisiopatologia , Isquemia/genética , Isquemia/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Músculo Esquelético/fisiopatologia , Doença Arterial Periférica/genética , Doença Arterial Periférica/fisiopatologia , Fosfolipases A2 Independentes de Cálcio/genética , Proteínas Supressoras de Tumor/genética , CaminhadaRESUMO
We have taken the first steps towards a complete reconstruction of the Mycobacterium tuberculosis regulatory network based on ChIP-Seq and combined this reconstruction with system-wide profiling of messenger RNAs, proteins, metabolites and lipids during hypoxia and re-aeration. Adaptations to hypoxia are thought to have a prominent role in M. tuberculosis pathogenesis. Using ChIP-Seq combined with expression data from the induction of the same factors, we have reconstructed a draft regulatory network based on 50 transcription factors. This network model revealed a direct interconnection between the hypoxic response, lipid catabolism, lipid anabolism and the production of cell wall lipids. As a validation of this model, in response to oxygen availability we observe substantial alterations in lipid content and changes in gene expression and metabolites in corresponding metabolic pathways. The regulatory network reveals transcription factors underlying these changes, allows us to computationally predict expression changes, and indicates that Rv0081 is a regulatory hub.
Assuntos
Redes Reguladoras de Genes , Hipóxia/genética , Redes e Vias Metabólicas/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Adaptação Fisiológica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Imunoprecipitação da Cromatina , Perfilação da Expressão Gênica , Redes Reguladoras de Genes/genética , Genômica , Hipóxia/metabolismo , Metabolismo dos Lipídeos/genética , Modelos Biológicos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/fisiologia , Oxigênio/farmacologia , Proteólise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Análise de Sequência de DNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tuberculose/metabolismo , Tuberculose/microbiologiaRESUMO
Cell growth and proliferation are tightly coupled to metabolism, and dissecting the signaling molecules which link these processes is an important step toward understanding development, regeneration, and cancer. The transcriptional regulator Yes-associated protein 1 (YAP) is a key regulator of liver size, development, and function. We now show that YAP can also suppress gluconeogenic gene expression. Yap deletion in primary hepatocytes potentiates the gluconeogenic gene response to glucagon and dexamethasone, whereas constitutively active YAP suppresses it. The effects of YAP are mediated by the transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator 1. YAP inhibits its ability to bind to and activate transcription from the promoters of its gluconeogenic targets, and the effects of YAP are blunted upon its knockdown. In vivo, constitutively active YAP lowers plasma glucose levels and increases liver size. CONCLUSION: YAP appears to reprogram cellular metabolism, diverting substrates away from the energy-consuming process of gluconeogenesis and toward the anabolic process of growth. (Hepatology 2017;66:2029-2041).
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação da Expressão Gênica , Gluconeogênese/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fosfoproteínas/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Proteínas de Ciclo Celular , Glucose-6-Fosfatase/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Cultura Primária de Células , Distribuição Aleatória , Fatores de Transcrição , Proteínas de Sinalização YAPRESUMO
Uterine leiomyomata (UL), the most common neoplasm in reproductive-age women, are classified into distinct genetic subgroups based on recurrent chromosome abnormalities. To develop a molecular signature of UL with t(12;14)(q14-q15;q23-q24), we took advantage of the multiple UL arising as independent clonal lesions within a single uterus. We compared genome-wide expression levels of t(12;14) UL to non-t(12;14) UL from each of nine women in a paired analysis, with each sample weighted for the percentage of t(12;14) cells to adjust for mosaicism with normal cells. This resulted in a transcriptional profile that confirmed HMGA2, known to be overexpressed in t(12;14) UL, as the most significantly altered gene. Pathway analysis of the differentially expressed genes showed significant association with cell proliferation, particularly G1/S checkpoint regulation. This is consistent with the known larger size of t(12;14) UL relative to karyotypically normal UL or to UL in the deletion 7q22 subgroup. Unsupervised hierarchical clustering demonstrated that patient variability is relatively dominant to the distinction of t(12;14) UL compared with non-t(12;14) UL or of t(12;14) UL compared with del(7q) UL. The paired design we employed is therefore important to produce an accurate t(12;14) UL-specific gene list by removing the confounding effects of genotype and environment. Interestingly, myometrium not only clustered away from the tumors, but generally separated based on associated t(12;14) versus del(7q) status. Nine genes were identified whose expression can distinguish the myometrium origin. This suggests an underlying constitutional genetic predisposition to these somatic changes which could potentially lead to improved personalized management and treatment.
Assuntos
Aberrações Cromossômicas , Heterogeneidade Genética , Predisposição Genética para Doença , Leiomioma/genética , Transcrição Gênica , Neoplasias Uterinas/genética , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Proteína HMGA2/genética , Proteína HMGA2/metabolismo , Humanos , Cariotipagem , Leiomioma/patologia , Miométrio/metabolismo , Miométrio/patologia , Neoplasias Uterinas/patologiaRESUMO
The filamentous fungus Neurospora crassa played a central role in the development of twentieth-century genetics, biochemistry and molecular biology, and continues to serve as a model organism for eukaryotic biology. Here, we have reconstructed a genome-scale model of its metabolism. This model consists of 836 metabolic genes, 257 pathways, 6 cellular compartments, and is supported by extensive manual curation of 491 literature citations. To aid our reconstruction, we developed three optimization-based algorithms, which together comprise Fast Automated Reconstruction of Metabolism (FARM). These algorithms are: LInear MEtabolite Dilution Flux Balance Analysis (limed-FBA), which predicts flux while linearly accounting for metabolite dilution; One-step functional Pruning (OnePrune), which removes blocked reactions with a single compact linear program; and Consistent Reproduction Of growth/no-growth Phenotype (CROP), which reconciles differences between in silico and experimental gene essentiality faster than previous approaches. Against an independent test set of more than 300 essential/non-essential genes that were not used to train the model, the model displays 93% sensitivity and specificity. We also used the model to simulate the biochemical genetics experiments originally performed on Neurospora by comprehensively predicting nutrient rescue of essential genes and synthetic lethal interactions, and we provide detailed pathway-based mechanistic explanations of our predictions. Our model provides a reliable computational framework for the integration and interpretation of ongoing experimental efforts in Neurospora, and we anticipate that our methods will substantially reduce the manual effort required to develop high-quality genome-scale metabolic models for other organisms.
Assuntos
Genoma Fúngico , Modelos Biológicos , Neurospora crassa/genética , AlgoritmosRESUMO
Activating brown adipose tissue (BAT) improves systemic metabolism, making it a promising target for metabolic syndrome. BAT is activated by 12,13-dihydroxy-9Z-octadecenoic acid (12,13-diHOME), which we previously identified to be inversely associated with BMI and which directly improves metabolism in multiple tissues. Here we profile plasma lipidomics from 83 people and test which lipids' association with BMI replicates in a concordant direction using our novel tool ScreenDMT, whose power and validity we demonstrate via mathematical proofs and simulations. We find that the linoleic acid diols 12,13-diHOME and 9,10-diHOME are both replicably inversely associated with BMI and mechanistically activate calcium influx in mouse brown and white adipocytes in vitro, which implicates this signaling pathway and 9,10-diHOME as candidate therapeutic targets. ScreenDMT can be applied to test directional mediation, directional replication, and qualitative interactions, such as identifying biomarkers whose association is shared (replication) or opposite (qualitative interaction) across diverse populations.
Assuntos
Índice de Massa Corporal , Cálcio , Animais , Camundongos , Humanos , Cálcio/metabolismo , Masculino , Adipócitos/metabolismo , Feminino , Tecido Adiposo Marrom/metabolismo , LipidômicaRESUMO
Wound research has typically been performed without regard for where the wounds are located on the body, despite well-known heterogeneities in physical and biological properties between different skin areas. The skin covering the palms and soles is highly specialized, and plantar ulcers are one of the most challenging and costly wound types to manage. Using primarily the porcine model, we show that plantar skin is molecularly and functionally more distinct from nonplantar skin than previously recognized, with unique gene and protein expression profiles, broad alterations in cellular functions, constitutive activation of many wound-associated phenotypes, and inherently delayed healing. This unusual physiology is likely to play a significant but underappreciated role in the pathogenesis of plantar ulcers as well as the last 25+ years of futility in therapy development efforts. By revealing this critical yet unrecognized pitfall, we hope to contribute to the development of more effective therapies for these devastating nonhealing wounds.
Assuntos
Fenótipo , Pele , Cicatrização , Animais , Cicatrização/fisiologia , Suínos , Pele/patologia , Pele/lesões , Pele/metabolismo , Modelos Animais de Doenças , Úlcera do Pé/fisiopatologia , Úlcera do Pé/patologia , Humanos , Feminino , Fenômenos Fisiológicos da Pele , PéRESUMO
OBJECTIVE: We aimed to identify metabolites associated with loss of glycemic control in youth-onset type 2 diabetes. RESEARCH DESIGN AND METHODS: We measured 480 metabolites in fasting plasma samples from the TODAY (Treatment Options for Type 2 Diabetes in Adolescents and Youth) study. Participants (N = 393; age 10-17 years) were randomly assigned to metformin, metformin plus rosiglitazone, or metformin plus lifestyle intervention. Additional metabolomic measurements after 36 months were obtained in 304 participants. Cox models were used to assess baseline metabolites, interaction of metabolites and treatment group, and change in metabolites (0-36 months), with loss of glycemic control adjusted for age, sex, race, treatment group, and BMI. Metabolite prediction models of glycemic failure were generated using elastic net regression and compared with clinical risk factors. RESULTS: Loss of glycemic control (HbA1c ≥8% or insulin therapy) occurred in 179 of 393 participants (mean 12.4 months). Baseline levels of 33 metabolites were associated with loss of glycemic control (q < 0.05). Associations of hexose and xanthurenic acid with treatment failure differed by treatment randomization; youths with higher baseline levels of these two compounds had a lower risk of treatment failure with metformin alone. For three metabolites, changes from 0 to 36 months were associated with loss of glycemic control (q < 0.05). Changes in d-gluconic acid and 1,5-AG/1-deoxyglucose, but not baseline levels of measured metabolites, predicted treatment failure better than changes in HbA1c or measures of ß-cell function. CONCLUSIONS: Metabolomics provides insight into circulating small molecules associated with loss of glycemic control and may highlight metabolic pathways contributing to treatment failure in youth-onset diabetes.
RESUMO
Insulin acts through the insulin receptor (IR) tyrosine kinase to exert its classical metabolic and mitogenic actions. Here, using receptors with either short or long deletion of the ß-subunit or mutation of the kinase active site (K1030R), we have uncovered a second, previously unrecognized IR signaling pathway that is intracellular domain-dependent, but ligand and tyrosine kinase-independent (LYK-I). These LYK-I actions of the IR are linked to changes in phosphorylation of a network of proteins involved in the regulation of extracellular matrix organization, cell cycle, ATM signaling and cellular senescence; and result in upregulation of expression of multiple extracellular matrix-related genes and proteins, down-regulation of immune/interferon-related genes and proteins, and increased sensitivity to apoptosis. Thus, in addition to classical ligand and tyrosine kinase-dependent (LYK-D) signaling, the IR regulates a second, ligand and tyrosine kinase-independent (LYK-I) pathway, which regulates the cellular machinery involved in senescence, matrix interaction and response to extrinsic challenges.
Assuntos
Apoptose , Divisão Celular , Senescência Celular , Proteínas Tirosina Quinases , Receptor de Insulina , Apoptose/genética , Divisão Celular/genética , Insulina/metabolismo , Ligantes , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Senescência Celular/genética , Humanos , Animais , CamundongosRESUMO
Pancreatic ß-cells are specialized for coupling glucose metabolism to insulin peptide production and secretion. Acute glucose exposure robustly and coordinately increases translation of proinsulin and proteins required for secretion of mature insulin peptide. By contrast, chronically elevated glucose levels that occur during diabetes impair ß-cell insulin secretion and have been shown experimentally to suppress insulin translation. Whether translation of other genes critical for insulin secretion are similarly downregulated by chronic high glucose is unknown. Here, we used high-throughput ribosome profiling and nascent proteomics in MIN6 insulinoma cells to elucidate the genome-wide impact of sustained high glucose on ß-cell mRNA translation. Prior to induction of ER stress or suppression of global translation, sustained high glucose suppressed glucose-stimulated insulin secretion and downregulated translation of not only insulin, but also of mRNAs related to insulin secretory granule formation, exocytosis, and metabolism-coupled insulin secretion. Translation of these mRNAs was also downregulated in primary rat and human islets following ex-vivo incubation with sustained high glucose and in an in vivo model of chronic mild hyperglycemia. Furthermore, translational downregulation decreased cellular abundance of these proteins. Our findings uncover a translational regulatory circuit during ß-cell glucose toxicity that impairs expression of proteins with critical roles in ß-cell function.
RESUMO
Pancreatic ß cells are specialized for coupling glucose metabolism to insulin peptide production and secretion. Acute glucose exposure robustly and coordinately increases translation of proinsulin and proteins required for secretion of mature insulin peptide. By contrast, chronically elevated glucose levels that occur during diabetes impair ß cell insulin secretion and have been shown experimentally to suppress insulin translation. Whether translation of other genes critical for insulin secretion is similarly downregulated by chronic high glucose is unknown. Here, we used high-throughput ribosome profiling and nascent proteomics in MIN6 insulinoma cells to elucidate the genome-wide impact of sustained high glucose on ß cell mRNA translation. Before induction of ER stress or suppression of global translation, sustained high glucose suppressed glucose-stimulated insulin secretion and downregulated translation of not only insulin, but also mRNAs related to insulin secretory granule formation, exocytosis, and metabolism-coupled insulin secretion. Translation of these mRNAs was also downregulated in primary rat and human islets following ex vivo incubation with sustained high glucose and in an in vivo model of chronic mild hyperglycemia. Furthermore, translational downregulation decreased cellular abundance of these proteins. Our study uncovered a translational regulatory circuit during ß cell glucose toxicity that impairs expression of proteins with critical roles in ß cell function.
Assuntos
Hiperglicemia , Células Secretoras de Insulina , Ilhotas Pancreáticas , Neoplasias Pancreáticas , Ratos , Humanos , Animais , Secreção de Insulina , RNA Mensageiro/metabolismo , Insulina/metabolismo , Hiperglicemia/genética , Hiperglicemia/metabolismo , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Peptídeos/metabolismo , Neoplasias Pancreáticas/metabolismo , Ilhotas Pancreáticas/metabolismoRESUMO
Insulin and IGF-1 receptors (IR and IGF1R) are highly homologous and share similar signaling systems, but each has a unique physiological role, with IR primarily regulating metabolic homeostasis and IGF1R regulating mitogenic control and growth. Here, we show that replacement of a single amino acid at position 973, just distal to the NPEY motif in the intracellular juxtamembrane region, from leucine, which is highly conserved in IRs, to phenylalanine, the highly conserved homologous residue in IGF1Rs, resulted in decreased IRS-1/PI3K/Akt/mTORC1 signaling and increased Shc/Gab1/MAPK cell cycle signaling. As a result, cells expressing L973F-IR exhibited decreased insulin-induced glucose uptake, increased cell growth, and impaired receptor internalization. Mice with knockin of the L973F-IR showed similar alterations in signaling in vivo, and this led to decreased insulin sensitivity, a modest increase in growth, and decreased weight gain when mice were challenged with a high-fat diet. Thus, leucine-973 in the juxtamembrane region of the IR acts as a crucial residue differentiating IR signaling from IGF1R signaling.