RESUMO
Lipids contribute to the structure, development, and function of healthy brains. Dysregulated lipid metabolism is linked to aging and diseased brains. However, our understanding of lipid metabolism in aging brains remains limited. Here we examined the brain lipidome of mice across their lifespan using untargeted lipidomics. Co-expression network analysis highlighted a progressive decrease in 3-sulfogalactosyl diacylglycerols (SGDGs) and SGDG pathway members, including the potential degradation products lyso-SGDGs. SGDGs show an age-related decline specifically in the central nervous system and are associated with myelination. We also found that an SGDG dramatically suppresses LPS-induced gene expression and release of pro-inflammatory cytokines from macrophages and microglia by acting on the NF-κB pathway. The detection of SGDGs in human and macaque brains establishes their evolutionary conservation. This work enhances interest in SGDGs regarding their roles in aging and inflammatory diseases and highlights the complexity of the brain lipidome and potential biological functions in aging.
Assuntos
Envelhecimento , Lipídeos , Animais , Humanos , Camundongos , Envelhecimento/genética , Anti-Inflamatórios , Encéfalo/metabolismo , Microglia/metabolismo , NF-kappa B/metabolismoRESUMO
Over the past decade, advances in genomics have identified thousands of additional protein-coding small open reading frames (smORFs) missed by traditional gene finding approaches. These smORFs encode peptides and small proteins, commonly termed micropeptides or microproteins. Several of these newly discovered microproteins have biological functions and operate through interactions with proteins and protein complexes within the cell. CYREN1 is a characterized microprotein that regulates double-strand break repair in mammalian cells through interaction with Ku70/80 heterodimer. Ku70/80 binds to and stabilizes double-strand breaks and recruits the machinery needed for nonhomologous end join repair. In this study, we examined the biochemical properties of CYREN1 to better understand and explain its cellular protein interactions. Our findings support that CYREN1 is an intrinsically disordered microprotein and this disordered structure allows it to enriches several proteins, including a newly discovered interaction with SF3B1 via a distinct short linear motif (SLiMs) on CYREN1. Since many microproteins are predicted to be disordered, CYREN1 is an exemplar of how microproteins interact with other proteins and reveals an unknown scaffolding function of this microprotein that may link NHEJ and splicing.
Assuntos
Peptídeos , Proteínas , Animais , Proteínas/genética , Peptídeos/genética , Fases de Leitura Aberta , Mamíferos/genética , MicropeptídeosRESUMO
Fatty acid esters of hydroxy fatty acids (FAHFAs) are a newly discovered class of signaling lipids with anti-inflammatory and anti-diabetic properties. However, the endogenous regulation of FAHFAs remains a pressing but unanswered question. Here, using MS-based FAHFA hydrolysis assays, LC-MS-based lipidomics analyses, and activity-based protein profiling, we found that androgen-induced gene 1 (AIG1) and androgen-dependent TFPI-regulating protein (ADTRP), two threonine hydrolases, control FAHFA levels in vivo in both genetic and pharmacologic mouse models. Tissues from mice lacking ADTRP (Adtrp-KO), or both AIG1 and ADTRP (DKO) had higher concentrations of FAHFAs particularly isomers with the ester bond at the 9th carbon due to decreased FAHFA hydrolysis activity. The levels of other lipid classes were unaltered indicating that AIG1 and ADTRP specifically hydrolyze FAHFAs. Complementing these genetic studies, we also identified a dual AIG1/ADTRP inhibitor, ABD-110207, which is active in vivo Acute treatment of WT mice with ABD-110207 resulted in elevated FAHFA levels, further supporting the notion that AIG1 and ADTRP activity control endogenous FAHFA levels. However, loss of AIG1/ADTRP did not mimic the changes associated with pharmacologically administered FAHFAs on extent of upregulation of FAHFA levels, glucose tolerance, or insulin sensitivity in mice, indicating that therapeutic strategies should weigh more on FAHFA administration. Together, these findings identify AIG1 and ADTRP as the first endogenous FAHFA hydrolases identified and provide critical genetic and chemical tools for further characterization of these enzymes and endogenous FAHFAs to unravel their physiological functions and roles in health and disease.
Assuntos
Esterases/metabolismo , Ésteres/química , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Proteínas de Membrana/metabolismo , Animais , Esterases/deficiência , Esterases/genética , Técnicas de Inativação de Genes , Hidrólise , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , CamundongosRESUMO
Recent technological advances led to the discovery of hundreds to thousands of peptides and small proteins (microproteins) encoded by small open reading frames (smORFs). Characterization of new microproteins demonstrates their role in fundamental biological processes and highlights the value in discovering and characterizing more microproteins. The elucidation of microprotein-protein interactions (MPIs) is useful for determining the biochemical and cellular roles of microproteins. In this study, we characterize the protein interaction partners of mitochondrial elongation factor 1 microprotein (MIEF1-MP) using a proximity labeling strategy that relies on APEX2. MIEF1-MP localizes to the mitochondrial matrix where it interacts with the mitochondrial ribosome (mitoribosome). Functional studies demonstrate that MIEF1-MP regulates mitochondrial translation via its binding to the mitoribosome. Loss of MIEF1-MP decreases the mitochondrial translation rate, while an elevated level of MIEF1-MP increases the translation rate. The identification of MIEF1-MP reveals a new gene involved in this process.
Assuntos
Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Fases de Leitura Aberta , Fatores de Alongamento de Peptídeos/metabolismo , Biossíntese de Proteínas , Sequência de Aminoácidos , Células HEK293 , Células HeLa , Humanos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Fatores de Alongamento de Peptídeos/genética , Homologia de SequênciaRESUMO
Microproteins are peptides and small proteins encoded by small open reading frames (smORFs). Newer technologies have led to the recent discovery of hundreds to thousands of new microproteins. The biological functions of a few microproteins have been elucidated, and these microproteins have fundamental roles in biology ranging from limb development to muscle function, highlighting the value of characterizing these molecules. The identification of microprotein-protein interactions (MPIs) has proven to be a successful approach to the functional characterization of these genes; however, traditional immunoprecipitation methods result in the enrichment of nonspecific interactions for microproteins. Here, we test and apply an in situ proximity tagging method that relies on an engineered ascorbate peroxidase 2 (APEX) to elucidate MPIs. The results demonstrate that APEX tagging is superior to traditional immunoprecipitation methods for microproteins. Furthermore, the application of APEX tagging to an uncharacterized microprotein called C11orf98 revealed that this microprotein interacts with nucleolar proteins nucleophosmin and nucleolin, demonstrating the ability of this approach to identify novel hypothesis-generating MPIs.
Assuntos
Ascorbato Peroxidases/metabolismo , Nucléolo Celular/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Engenharia de Proteínas , Proteômica/métodos , Proteínas de Ligação a RNA/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Ascorbato Peroxidases/química , Ascorbato Peroxidases/genética , Sequência Conservada , Células HEK293 , Células HeLa , Hemaglutininas/química , Hemaglutininas/genética , Hemaglutininas/metabolismo , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Proteínas Nucleares/química , Proteínas Nucleares/genética , Nucleofosmina , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Domínios e Motivos de Interação entre Proteínas , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , NucleolinaRESUMO
We recently discovered a structurally novel class of endogenous lipids, branched palmitic acid esters of hydroxy stearic acids (PAHSAs), with beneficial metabolic and anti-inflammatory effects. We tested whether PAHSAs protect against colitis, which is a chronic inflammatory disease driven predominantly by defects in the innate mucosal barrier and adaptive immune system. There is an unmet clinical need for safe and well tolerated oral therapeutics with direct anti-inflammatory effects. Wild-type mice were pretreated orally with vehicle or 5-PAHSA (10 mg/kg) and 9-PAHSA (5 mg/kg) once daily for 3 days, followed by 10 days of either 0% or 2% dextran sulfate sodium water with continued vehicle or PAHSA treatment. The colon was collected for histopathology, gene expression, and flow cytometry. Intestinal crypt fractions were prepared for ex vivo bactericidal assays. Bone marrow-derived dendritic cells pretreated with vehicle or PAHSA and splenic CD4+ T cells from syngeneic mice were co-cultured to assess antigen presentation and T cell activation in response to LPS. PAHSA treatment prevented weight loss, improved colitis scores (stool consistency, hematochezia, and mouse appearance), and augmented intestinal crypt Paneth cell bactericidal potency via a mechanism that may involve GPR120. In vitro, PAHSAs attenuated dendritic cell activation and subsequent T cell proliferation and Th1 polarization. The anti-inflammatory effects of PAHSAs in vivo resulted in reduced colonic T cell activation and pro-inflammatory cytokine and chemokine expression. These anti-inflammatory effects appear to be partially GPR120-dependent. We conclude that PAHSA treatment regulates innate and adaptive immune responses to prevent mucosal damage and protect against colitis. Thus, PAHSAs may be a novel treatment for colitis and related inflammation-driven diseases.
Assuntos
Imunidade Adaptativa/imunologia , Colite/tratamento farmacológico , Ácidos Graxos/farmacologia , Imunidade Inata/efeitos dos fármacos , Imunidade nas Mucosas/efeitos dos fármacos , Celulas de Paneth/imunologia , Células Th1/imunologia , Animais , Colite/induzido quimicamente , Colite/imunologia , Colite/patologia , Sulfato de Dextrana/efeitos adversos , Sulfato de Dextrana/farmacologia , Masculino , Camundongos , Celulas de Paneth/patologia , Receptores Acoplados a Proteínas G/imunologia , Células Th1/patologiaRESUMO
Apolipoprotein E (ApoE) belongs to a large class of proteins that solubilize lipids for physiological transport. Humans have three different APOE alleles, APOE ε2, APOE ε3, and APOE ε4, and genetic studies identified ApoE4 as the strongest genetic risk factor for Alzheimer's disease (AD). People who are homozygous for ApoE4 (i.e., ApoE4/E4) are an order of magnitude more likely to develop late-onset AD (LOAD) than ApoE3/E3 carriers. Several differences between ApoE3 and ApoE4 may contribute to AD including the observation that ApoE4 is degraded to a greater extent than ApoE3 in the human brain. Experiments with high-temperature requirement serine peptidase A1 (HtrA1), which is found in the nervous system, demonstrate that HtrA1 is an allele-selective ApoE-degrading enzyme that degrades ApoE4 more quickly than ApoE3. This activity is specific to HtrA1, as similar assays with HtrA2 showed minimal ApoE4 proteolysis and trypsin had no preference between ApoE4 and ApoE3. HtrA1 has also been reported to cleave the tau protein (Tau) and the amyloid protein precursor (APP) to hinder the formation of toxic amyloid deposits associated with AD. Competition assays with ApoE4, ApoE3, and Tau revealed that ApoE4 inhibits Tau degradation. Thus, the identification of ApoE4 as an in vitro HtrA1 substrate suggests a potential biochemical mechanism that links ApoE4 regulation of AD proteins such as Tau.
Assuntos
Alelos , Apolipoproteínas E/genética , Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Proteólise , Células HEK293 , Serina Peptidase 1 de Requerimento de Alta Temperatura A/química , Humanos , Modelos Moleculares , Conformação ProteicaRESUMO
The corticotropin-releasing factor (CRF) peptide family comprises the mammalian peptides CRF and the urocortins as well as frog skin sauvagine and fish urophyseal urotensin. Advances in understanding the roles of the CRF ligand family and associated receptors have often relied on radioreceptor assays using labeled CRF ligands. These assays depend on stable, high-affinity CRF analogs that can be labeled, purified, and chemically characterized. Analogs of several of the native peptides have been used in this context, most prominently including sauvagine from the frog Phyllomedusa sauvageii (PS-Svg). Because each of these affords both advantages and disadvantages, new analogs with superior properties would be welcome. We find that a sauvagine-like peptide recently isolated from a different frog species, Pachymedusa dacnicolor (PD-Svg), is a high-affinity agonist whose radioiodinated analog, [(125)ITyr(0)-Glu(1), Nle(17)]-PD-Svg, exhibits improved biochemical properties over those of earlier iodinated agonists. Specifically, the PD-Svg radioligand binds both CRF receptors with comparably high affinity as its PS-Svg counterpart, but detects a greater number of sites on both type 1 and type 2 receptors. PD-Svg is also â¼10 times more potent at stimulating cAMP accumulation in cells expressing the native receptors. Autoradiographic localization using the PD-Svg radioligand shows robust specific binding to rodent brain and peripheral tissues that identifies consensus CRF receptor-expressing sites in a greater number and/or with greater sensitivity than its PS-Svg counterpart. We suggest that labeled analogs of PD-Svg may be useful tools for biochemical, structural, pharmacological, and anatomic studies of CRF receptors.
Assuntos
Proteínas de Anfíbios/metabolismo , Anuros , Hormônios Peptídicos/metabolismo , Ensaio Radioligante/métodos , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Sequência de Aminoácidos , Proteínas de Anfíbios/química , Animais , Linhagem Celular , Humanos , Marcação por Isótopo , Cinética , Ligantes , Camundongos , Dados de Sequência Molecular , Hormônios Peptídicos/química , Transporte Proteico , Ratos , Receptores de Hormônio Liberador da Corticotropina/químicaRESUMO
BACKGROUND: Insulin producing beta cell and glucagon producing alpha cells are colocalized in pancreatic islets in an arrangement that facilitates the coordinated release of the two principal hormones that regulate glucose homeostasis and prevent both hypoglycemia and diabetes. However, this intricate organization has also complicated the determination of the cellular source(s) of the expression of genes that are detected in the islet. This reflects a significant gap in our understanding of mouse islet physiology, which reduces the effectiveness by which mice model human islet disease. RESULTS: To overcome this challenge, we generated a bitransgenic reporter mouse that faithfully labels all beta and alpha cells in mouse islets to enable FACS-based purification and the generation of comprehensive transcriptomes of both populations. This facilitates systematic comparison across thousands of genes between the two major endocrine cell types of the islets of Langerhans whose principal hormones are of cardinal importance for glucose homeostasis. Our data leveraged against similar data for human beta cells reveal a core common beta cell transcriptome of 9900+ genes. Against the backdrop of overall similar beta cell transcriptomes, we describe marked differences in the repertoire of receptors and long non-coding RNAs between mouse and human beta cells. CONCLUSIONS: The comprehensive mouse alpha and beta cell transcriptomes complemented by the comparison of the global (dis)similarities between mouse and human beta cells represent invaluable resources to boost the accuracy by which rodent models offer guidance in finding cures for human diabetes.
Assuntos
Células Secretoras de Insulina/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Citometria de Fluxo , Biblioteca Gênica , Glucagon/genética , Glucagon/metabolismo , Células Secretoras de Glucagon/citologia , Células Secretoras de Glucagon/metabolismo , Humanos , Células Secretoras de Insulina/citologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , RNA/genética , RNA/metabolismo , RNA Longo não Codificante/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Análise de Sequência de RNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma , Proteína Vermelha FluorescenteRESUMO
Peptide hormones are key physiological regulators, and many would make terrific drugs; however, the therapeutic use of peptides is limited by poor metabolism including rapid proteolysis. To develop novel proteolysis-resistant peptide hormone analogs, we utilize a strategy that relies on data from simple mass spectrometry experiments to guide the chemical synthesis of proteolysis-resistant analogs (i.e., data-driven synthesis). Application of this strategy to oxyntomodulin (OXM), a peptide hormone that stimulates insulin secretion from islets and lowers blood glucose in vivo, defined the OXM cleavage site in serum, and this information was used to synthesize a proteolysis-resistant OXM analog (prOXM). prOXM and OXM have similar activity in binding and glucose stimulated-insulin secretion assays. Furthermore, prOXM is also active in vivo. prOXM reduces basal glucose levels and improves glucose tolerance in mice. The discovery of prOXM suggests that proteolysis-resistant variants of other important peptide hormones can also be found using this strategy to increase the number of candidate therapeutic peptides.
Assuntos
Hormônios Peptídicos/síntese química , Proteólise , Sequência de Aminoácidos , Animais , Glicemia/metabolismo , Técnicas de Química Sintética , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Hormônios Peptídicos/química , Hormônios Peptídicos/metabolismo , Hormônios Peptídicos/farmacologiaRESUMO
Pancreatic alpha cell activity and glucagon secretion lower as glucose levels increase. While part of the decrease is regulated by glucose itself, paracrine signaling by their neighboring beta and delta cells also plays an important role. Somatostatin from delta cells is an important local inhibitor of alpha cells at high glucose. Additionally, urocortin 3 (UCN3) is a hormone that is co-released from beta cells with insulin and acts locally to potentiate somatostatin secretion from delta cells. UCN3 thus inhibits insulin secretion via a negative feedback loop with delta cells, but its role with respect to alpha cells and glucagon secretion is not understood. We hypothesize that the somatostatin-driven glucagon inhibition at high glucose is regulated in part by UCN3 from beta cells. Here, we use a combination of live functional Ca2+ and cAMP imaging as well as direct glucagon secretion measurement, all from alpha cells in intact mouse islets, to determine the contributions of UCN3 to alpha cell behavior. Exogenous UCN3 treatment decreased alpha cell Ca2+ and cAMP levels and inhibited glucagon release. Blocking endogenous UCN3 signaling increased alpha cell Ca2+ by 26.8 ± 7.6%, but this did not result in increased glucagon release at high glucose. Furthermore, constitutive deletion of Ucn3 did not increase Ca2+ activity or glucagon secretion relative to controls. UCN3 is thus capable of inhibiting mouse alpha cells, but, given the subtle effects of endogenous UCN3 signaling on alpha cells, we propose that UCN3-driven somatostatin may serve to regulate local paracrine glucagon levels in the islet instead of inhibiting gross systemic glucagon release.
Assuntos
Células Secretoras de Glucagon , Glucagon , Comunicação Parácrina , Urocortinas , Animais , Urocortinas/metabolismo , Urocortinas/genética , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Glucagon/efeitos dos fármacos , Camundongos , Glucagon/metabolismo , Glucose/metabolismo , Cálcio/metabolismo , Masculino , Camundongos Endogâmicos C57BL , AMP Cíclico/metabolismo , Somatostatina/farmacologia , Somatostatina/metabolismoRESUMO
Corticotropin-releasing factor (CRF), originally characterized as the principal neuroregulator of the hypothalamus-pituitary-adrenal axis, has broad central and peripheral distribution and actions. We demonstrate the presence of CRF receptor type 1 (CRFR1) on primary beta cells and show that activation of pancreatic CRFR1 promotes insulin secretion, thus contributing to the restoration of normoglycemic equilibrium. Stimulation of pancreatic CRFR1 initiates a cAMP response that promotes insulin secretion in vitro and in vivo and leads to the phosphorylation of cAMP response element binding and the induction of the expression of several immediate-early genes. Thus, the insulinotropic actions of pancreatic CRFR1 oppose the activation of CRFR1 on anterior pituitary corticotropes, leading to the release of glucocorticoids that functionally antagonize the actions of insulin. Stimulation of the MIN6 insulinoma line and primary rat islets with CRF also activates the MAPK signaling cascade leading to rapid phosphorylation of Erk1/2 in response to CRFR1-selective ligands, which induce proliferation in primary rat neonatal beta cells. Importantly, CRFR1 stimulates insulin secretion only during conditions of intermediate to high ambient glucose, and the CRFR1-dependent phosphorylation of Erk1/2 is greater with elevated glucose concentrations. This response is reminiscent of the actions of the incretins, which potentiate insulin secretion only during elevated glucose conditions. The presence of CRFR1 on beta cells adds another layer of complexity to the intricate network of paracrine and autocrine factors and their cognate receptors whose coordinated efforts can dictate islet hormone output and regulate beta cell proliferation.
Assuntos
Glucose/farmacologia , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/fisiologia , Insulina/metabolismo , Receptores de Hormônio Liberador da Corticotropina/genética , Adrenalectomia , Animais , Divisão Celular , Linhagem Celular Tumoral , AMP Cíclico/metabolismo , DNA Complementar/genética , Citometria de Fluxo , Teste de Tolerância a Glucose , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Insulinoma , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Período Pós-Prandial , Ratos , Receptores de Hormônio Liberador da Corticotropina/deficiênciaRESUMO
Cellular homeostasis relies on having dedicated and coordinated responses to a variety of stresses. The accumulation of unfolded proteins in the endoplasmic reticulum (ER) is a common stress that triggers a conserved pathway called the unfolded protein response (UPR) that mitigates damage, and dysregulation of UPR underlies several debilitating diseases. Here, we discover that a previously uncharacterized 54-amino acid microprotein PIGBOS regulates UPR. PIGBOS localizes to the mitochondrial outer membrane where it interacts with the ER protein CLCC1 at ER-mitochondria contact sites. Functional studies reveal that the loss of PIGBOS leads to heightened UPR and increased cell death. The characterization of PIGBOS reveals an undiscovered role for a mitochondrial protein, in this case a microprotein, in the regulation of UPR originating in the ER. This study demonstrates microproteins to be an unappreciated class of genes that are critical for inter-organelle communication, homeostasis, and cell survival.
Assuntos
Canais de Cloreto/metabolismo , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Proteínas Mitocondriais/metabolismo , Resposta a Proteínas não Dobradas , Animais , Células COS , Morte Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Membranas Mitocondriais/metabolismo , Mapas de Interação de Proteínas , Coelhos , RatosRESUMO
Pancreatic α cells retain considerable plasticity and can, under the right circumstances, transdifferentiate into functionally mature ß cells. In search of a targetable mechanistic basis, a recent paper suggested that the widely used anti-malaria drug artemether suppresses the α cell transcription factor Arx to promote transdifferentiation into ß cells. However, key initial experiments in this paper were carried out in islet cell lines, and most subsequent validation experiments implied transdifferentiation without direct demonstration of α to ß cell conversion. Indeed, we find no evidence that artemether promotes transdifferentiation of primary α cells into ß cells. Moreover, artemether reduces Ins2 expression in primary ß cells >100-fold, suppresses glucose uptake, and abrogates ß cell calcium responses and insulin secretion in response to glucose. Our observations suggest that artemether induces general islet endocrine cell dedifferentiation and call into question the utility of artemisinins to promote α to ß cell transdifferentiation in treating diabetes.
Assuntos
Artemeter/farmacologia , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Animais , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Transdiferenciação Celular , Células Cultivadas , Células Secretoras de Glucagon/efeitos dos fármacos , Glucose/metabolismo , Proteínas de Homeodomínio/metabolismo , Insulina/metabolismo , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Fatores de Transcrição/metabolismoRESUMO
Palmitic acid hydroxystearic acids (PAHSAs) are endogenous lipids with anti-diabetic and anti-inflammatory effects. PAHSA levels are reduced in serum and adipose tissue of insulin-resistant people and high-fat diet (HFD)-fed mice. Here, we investigated whether chronic PAHSA treatment enhances insulin sensitivity and which receptors mediate PAHSA effects. Chronic PAHSA administration in chow- and HFD-fed mice raises serum and tissue PAHSA levels â¼1.4- to 3-fold. This improves insulin sensitivity and glucose tolerance without altering body weight. PAHSA administration in chow-fed, but not HFD-fed, mice augments insulin and glucagon-like peptide (GLP-1) secretion. PAHSAs are selective agonists for GPR40, increasing Ca+2 flux, but not intracellular cyclic AMP. Blocking GPR40 reverses improvements in glucose tolerance and insulin sensitivity in PAHSA-treated chow- and HFD-fed mice and directly inhibits PAHSA augmentation of glucose-stimulated insulin secretion in human islets. In contrast, GLP-1 receptor blockade in PAHSA-treated chow-fed mice reduces PAHSA effects on glucose tolerance, but not on insulin sensitivity. Thus, PAHSAs activate GPR40, which is involved in their beneficial metabolic effects.
Assuntos
Glucose/metabolismo , Homeostase , Ácido Palmítico/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Ácidos Esteáricos/farmacologia , Adiposidade/efeitos dos fármacos , Animais , Ingestão de Alimentos/efeitos dos fármacos , Células HEK293 , Homeostase/efeitos dos fármacos , Humanos , Inflamação/patologia , Resistência à Insulina , Camundongos Endogâmicos C57BLRESUMO
Postnatal maintenance or regeneration of pancreatic beta cells is considered to occur exclusively via the replication of existing beta cells, but clinically meaningful restoration of human beta cell mass by proliferation has never been achieved. We discovered a population of immature beta cells that is present throughout life and forms from non-beta precursors at a specialized micro-environment or "neogenic niche" at the islet periphery. These cells express insulin, but lack other key beta cell markers, and are transcriptionally immature, incapable of sensing glucose, and unable to support calcium influx. They constitute an intermediate stage in the transdifferentiation of alpha cells to cells that are functionally indistinguishable from conventional beta cells. We thus identified a lifelong source of new beta cells at a specialized site within healthy islets. By comparing co-existing immature and mature beta cells within healthy islets, we stand to learn how to mature insulin-expressing cells into functional beta cells.
Assuntos
Envelhecimento/fisiologia , Microambiente Celular , Células Secretoras de Insulina/citologia , Adulto , Diferenciação Celular/genética , Transdiferenciação Celular , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Perfilação da Expressão Gênica , Glucagon/metabolismo , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Glucagon/patologia , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Doadores de Tecidos , Transcrição Gênica , Urocortinas/metabolismoRESUMO
Cocaine- and amphetamine-regulated transcript (CART) is a highly expressed peptide implicated in the regulation of feeding, reward and reinforcement, and stress-related behaviors. CART has been localized to discrete cell populations in the brain, gut, adrenal gland, and pancreas. In contrast, CART-producing cell types in the pituitary gland remain ill defined. In the present study, double-label immunohistochemistry, employing a high-affinity antiserum we generated against CART-(62-102), was used to identify CART-producing cells in the pituitary gland. In the anterior pituitary, the majority of CART immunoreactivity (-ir) was localized in lactotropes; minor populations of CART-ir cells were identified as somatotropes and corticotropes. In the posterior pituitary, CART-ir extensively colocalized with oxytocin-containing fibers; in contrast, only a few vasopressin fibers contained CART-ir. As expected, CART colocalized with oxytocin in magnocellular neurons of the supraoptic nucleus. The effects of bromocriptine, a potent dopamine receptor agonist, were examined to determine whether CART mRNA expression and protein release are regulated in a similar fashion as prolactin. Similar to prolactin, CART mRNA expression and protein release were significantly decreased after bromocriptine treatment of dispersed rat anterior pituitary cells in culture. To explore the putative physiological role of pituitary CART, we compared levels of CART mRNA expression in lactating and nonlactating female rats. CART mRNA levels were significantly increased in the anterior pituitary and supraoptic nucleus of lactating rats. Furthermore, levels of CART in the systemic circulation were significantly elevated at the onset of lactation, peaked on d 10 of lactation and returned to baseline values 10 d after pups were weaned. The current study describes the cellular localization and regulation of CART expression and protein release from the rat pituitary gland. These findings suggest a putative role for CART in lactation.
Assuntos
Regulação da Expressão Gênica , Lactação , Proteínas do Tecido Nervoso/biossíntese , Hipófise/metabolismo , Anfetaminas/farmacologia , Animais , Bromocriptina/farmacologia , Células Cultivadas , Cromatografia , Cromatografia em Gel , Cocaína/farmacologia , Feminino , Hipotálamo/metabolismo , Immunoblotting , Imuno-Histoquímica , Hibridização In Situ , Masculino , Modelos Estatísticos , Proteínas do Tecido Nervoso/fisiologia , Neurônios/metabolismo , Ocitocina/metabolismo , RNA Mensageiro/metabolismo , Radioimunoensaio , Ratos , Ratos Sprague-Dawley , Distribuição TecidualRESUMO
OBJECTIVE: Complex local crosstalk amongst endocrine cells within the islet ensures tight coordination of their endocrine output. This is illustrated by the recent demonstration that the negative feedback control by delta cells within pancreatic islets determines the homeostatic set-point for plasma glucose during mouse postnatal development. However, the close association of islet endocrine cells that facilitates paracrine crosstalk also complicates the distinction between effects mediated directly on beta cells from indirect effects mediated via local intermediates, such as somatostatin from delta cells. METHODS: To resolve this problem, we generated reporter mice that allow collection of pure pancreatic delta cells along with alpha and beta cells from the same islets and generated comprehensive transcriptomes for each islet endocrine cell type. These transcriptomes afford an unparalleled view of the receptors expressed by delta, alpha and beta cells, and allow the prediction of which signal targets which endocrine cell type with great accuracy. RESULTS: From these transcriptomes, we discovered that the ghrelin receptor is expressed exclusively by delta cells within the islet, which was confirmed by fluorescent in situ hybridization and qPCR. Indeed, ghrelin increases intracellular calcium in delta cells in intact mouse islets, measured by GCaMP6 and robustly potentiates glucose-stimulated somatostatin secretion on mouse and human islets in both static and perfusion assays. In contrast, des-acyl-ghrelin at the same dose had no effect on somatostatin secretion and did not block the actions of ghrelin. CONCLUSIONS: These results offer a straightforward explanation for the well-known insulinostatic actions of ghrelin. Rather than engaging beta cells directly, ghrelin engages delta cells to promote local inhibitory feedback that attenuates insulin release. These findings illustrate the power of our approach to resolve some of the long-standing conundrums with regard to the rich feedback that occurs within the islet that is integral to islet physiology and therefore highly relevant to diabetes.
RESUMO
The peptide hormone urocortin3 (Ucn3) is abundantly expressed by mature beta cells, yet its physiological role is unknown. Here we demonstrate that Ucn3 is stored and co-released with insulin and potentiates glucose-stimulated somatostatin secretion via cognate receptors on delta cells. Further, we found that islets lacking endogenous Ucn3 have fewer delta cells, reduced somatostatin content, impaired somatostatin secretion, and exaggerated insulin release, and that these defects are rectified by treatment with synthetic Ucn3 in vitro. Our observations indicate that the paracrine actions of Ucn3 activate a negative feedback loop that promotes somatostatin release to ensure the timely reduction of insulin secretion upon normalization of plasma glucose. Moreover, Ucn3 is markedly depleted from beta cells in mouse and macaque models of diabetes and in human diabetic islets. This suggests that Ucn3 is a key contributor to stable glycemic control, whose reduction during diabetes aggravates glycemic volatility and contributes to the pathophysiology of this disease.
Assuntos
Retroalimentação Fisiológica , Insulina/metabolismo , Somatostatina/metabolismo , Urocortinas/metabolismo , Adolescente , Adulto , Idoso , Animais , Criança , Pré-Escolar , Diabetes Mellitus/genética , Diabetes Mellitus/patologia , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Hiperglicemia/genética , Hiperglicemia/patologia , Lactente , Recém-Nascido , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Macaca , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Modelos Biológicos , Comunicação Parácrina , Doadores de Tecidos , Transcriptoma/genética , Urocortinas/deficiência , Adulto JovemRESUMO
Activins are dimeric proteins that stimulate the synthesis and secretion of pituitary FSH by interacting with two classes of receptors, type I and type II, to initiate their intracellular signaling cascade. The extracellular domain of type II activin receptor (ActRII-ECD) contains all structural determinants sufficient for high affinity ligand binding. A soluble recombinant ActRII-ECD has been reported to attenuate FSH secretion from cultured rat anterior pituitary cells in response to exogenous activin A or endogenous activin B. Follistatin is a binding protein that acts as an extracellular factor to bind and inactivate activin. We constructed adenoviral vectors able to mediate expression of follistatin 288 (AdexCAFS288) and ActRII-ECD (AdexCAECD) and tested their biological activities both in vitro and in vivo. The data show that adenovirus-mediated overexpression of either ActRII-ECD or follistatin was able to attenuate FSH secretion by cultured rat anterior pituitary cells. However, AdexCAFS288 overexpression of follistatin was more effective than adenovirus-mediated overexpression of ActRII-ECD. In vivo, a single ip injection of AdexCAFS288 induced the expression of high levels of follistatin and resulted in the suppression of serum FSH levels in castrated male rats for up to 12 d postinjection. Infection with AdexCAFS288 had no effect on LH secretion in vitro or in vivo, demonstrating its selectivity. In conclusion, the results demonstrate the effectiveness of adenovirus-mediated overexpression of follistatin and ActRII-ECD to regulate FSH secretion and the potential of using this strategy as a tool to further define the critical role of activin/inhibin/follistatin circuitry in the modulation of the reproductive system.