RESUMEN
Syntrophins are a family of proteins forming membrane-anchored scaffolds and serving as adaptors for various transmembrane and intracellular signaling molecules. To understand the physiological roles of ß1 syntrophin, one of the least characterized members, we generated mouse models to eliminate ß1 syntrophin specifically in the endocrine or exocrine pancreas. ß1 syntrophin is dispensable for the morphology and function of insulin-producing ß cells. However, mice with ß1 syntrophin deletion in exocrine acinar cells exhibit increased severity of cerulein-induced acute pancreatitis. Reduced expression of cystic fibrosis transmembrane conductance regulator and dilation of acinar lumen are potential predisposition factors. During the disease progression, a relative lack of autophagy is associated with deficiencies in both actin assembly and endoplasmic reticulum nucleation. Our findings reveal, for the first time, that ß1 syntrophin is a critical regulator of actin cytoskeleton and autophagy in pancreatic acinar cells and is potently protective against cerulein-induced acute pancreatitis.
Asunto(s)
Autofagia , Ceruletida/toxicidad , Proteínas Asociadas a la Distrofina/fisiología , Pancreatitis/prevención & control , Sustancias Protectoras , Células Acinares/efectos de los fármacos , Células Acinares/metabolismo , Animales , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Páncreas/efectos de los fármacos , Páncreas/metabolismo , Pancreatitis/inducido químicamente , Pancreatitis/metabolismo , Pancreatitis/patologíaRESUMEN
Regulation of neurotransmitter release during presynaptic plasticity underlies varied forms of information processing in the brain. Munc13s play essential roles in release via their conserved C-terminal region, which contains a MUN domain involved SNARE complex assembly, and control multiple presynaptic plasticity processes. Munc13s also have a variable N-terminal region, which in Munc13-1 includes a calmodulin binding (CaMb) domain involved in short-term plasticity and a C2A domain that forms an inhibitory homodimer. The C2A domain is activated by forming a heterodimer with the zinc-finger domain of αRIMs, providing a link to αRIM-dependent short- and long-term plasticity. However, it is unknown how the functions of the N- and C-terminal regions are integrated, in part because of the difficulty of purifying Munc13-1 fragments containing both regions. We describe for the first time the purification of a Munc13-1 fragment spanning its entire sequence except for a flexible region between the C2A and CaMb domains. We show that this fragment is much less active than the Munc13-1 C-terminal region in liposome fusion assays and that its activity is strongly enhanced by the RIM2α zinc-finger domain together with calmodulin. NMR experiments show that the C2A and CaMb domains bind to the MUN domain and that these interactions are relieved by the RIM2α ZF domain and calmodulin, respectively. These results suggest a model whereby Munc13-1 activity in promoting SNARE complex assembly and neurotransmitter release are inhibited by interactions of the C2A and CaMb domains with the MUN domain that are relieved by αRIMs and calmodulin.
RESUMEN
Regulation of neurotransmitter release during presynaptic plasticity underlies varied forms of information processing in the brain. Munc13s play essential roles in release via their conserved C-terminal region, which contains a MUN domain involved in SNARE complex assembly, and controls multiple presynaptic plasticity processes. Munc13s also have a variable N-terminal region, which in Munc13-1 includes a calmodulin binding (CaMb) domain involved in short-term plasticity and a C2A domain that forms an inhibitory homodimer. The C2A domain is activated by forming a heterodimer with the zinc-finger domain of αRIMs, providing a link to αRIM-dependent short- and long-term plasticity. However, it is unknown how the functions of the N- and C-terminal regions are integrated, in part because of the difficulty of purifying Munc13-1 fragments containing both regions. We describe for the first time the purification of a Munc13-1 fragment spanning its entire sequence except for a flexible region between the C2A and CaMb domains. We show that this fragment is much less active than the Munc13-1 C-terminal region in liposome fusion assays and that its activity is strongly enhanced by the RIM2α zinc-finger domain together with calmodulin. NMR experiments show that the C2A and CaMb domains bind to the MUN domain and that these interactions are relieved by the RIM2α ZF domain and calmodulin, respectively. These results suggest a model whereby Munc13-1 activity in promoting SNARE complex assembly and neurotransmitter release are inhibited by interactions of the C2A and CaMb domains with the MUN domain that are relieved by αRIMs and calmodulin.
Asunto(s)
Calmodulina , Proteínas del Tejido Nervioso , Calmodulina/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Neurotransmisores , Proteínas SNARE/metabolismo , Zinc/metabolismo , HumanosRESUMEN
Neurotransmitter release is triggered in microseconds by Ca2+-binding to the Synaptotagmin-1 C2 domains and by SNARE complexes that form four-helix bundles between synaptic vesicles and plasma membranes, but the coupling mechanism between Ca2+-sensing and membrane fusion is unknown. Release requires extension of SNARE helices into juxtamembrane linkers that precede transmembrane regions (linker zippering) and binding of the Synaptotagmin-1 C2B domain to SNARE complexes through a 'primary interface' comprising two regions (I and II). The Synaptotagmin-1 Ca2+-binding loops were believed to accelerate membrane fusion by inducing membrane curvature, perturbing lipid bilayers or helping bridge the membranes, but SNARE complex binding orients the Ca2+-binding loops away from the fusion site, hindering these putative activities. Molecular dynamics simulations now suggest that Synaptotagmin-1 C2 domains near the site of fusion hinder SNARE action, providing an explanation for this paradox and arguing against previous models of Sytnaptotagmin-1 action. NMR experiments reveal that binding of C2B domain arginines to SNARE acidic residues at region II remains after disruption of region I. These results and fluorescence resonance energy transfer assays, together with previous data, suggest that Ca2+ causes reorientation of the C2B domain on the membrane and dissociation from the SNAREs at region I but not region II. Based on these results and molecular modeling, we propose that Synaptotagmin-1 acts as a lever that pulls the SNARE complex when Ca2+ causes reorientation of the C2B domain, facilitating linker zippering and fast membrane fusion. This hypothesis is supported by the electrophysiological data described in the accompanying paper.
RESUMEN
G protein-coupled receptor (GPCR) pathways control glucose and fatty acid metabolism and the onset of obesity and diabetes. Regulators of G protein signaling (RGS) are GTPase-activating proteins (GAPs) for G(i) and G(q) α-subunits that control the intensity and duration of GPCR signaling. Herein we determined the role of Rgs16 in GPCR regulation of liver metabolism. Rgs16 is expressed during the last few hours of the daily fast in periportal hepatocytes, the oxygen-rich zone of the liver where lipolysis and gluconeogenesis predominate. Rgs16 knock-out mice had elevated expression of fatty acid oxidation genes in liver, higher rates of fatty acid oxidation in liver extracts, and higher plasma ß-ketone levels compared with wild type mice. By contrast, transgenic mice that overexpressed RGS16 protein specifically in liver exhibited reciprocal phenotypes as well as low blood glucose levels compared with wild type littermates and fatty liver after overnight fasting. The transcription factor carbohydrate response element-binding protein (ChREBP), which induces fatty acid synthesis genes in response to high carbohydrate feeding, was unexpectedly required during fasting for maximal Rgs16 transcription in liver and in cultured primary hepatocytes during gluconeogenesis. Thus, RGS16 provides a signaling mechanism for glucose production to inhibit GPCR-stimulated fatty acid oxidation in hepatocytes.
Asunto(s)
Ácidos Grasos/metabolismo , Proteínas Nucleares/fisiología , Proteínas RGS/fisiología , Factores de Transcripción/fisiología , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Ácidos Grasos/biosíntesis , Ácidos Grasos/genética , Gluconeogénesis , Glucosa/biosíntesis , Glucosa/fisiología , Hepatocitos/metabolismo , Hígado/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Oxidación-Reducción , Receptores Acoplados a Proteínas G/metabolismo , Transcripción GenéticaRESUMEN
Cl(-) channels in the apical membrane of biliary epithelial cells (BECs) provide the driving force for ductular bile formation. Although a cystic fibrosis transmembrane conductance regulator has been identified in BECs and contributes to secretion via secretin binding basolateral receptors and increasing [cAMP](i), an alternate Cl(-) secretory pathway has been identified that is activated via nucleotides (ATP, UTP) binding apical P2 receptors and increasing [Ca(2+)](i). The molecular identity of this Ca(2+)-activated Cl(-) channel is unknown. The present studies in human, mouse, and rat BECs provide evidence that TMEM16A is the operative channel and contributes to Ca(2+)-activated Cl(-) secretion in response to extracellular nucleotides. Furthermore, Cl(-) currents measured from BECs isolated from distinct areas of intrahepatic bile ducts revealed important functional differences. Large BECs, but not small BECs, exhibit cAMP-stimulated Cl(-) currents. However, both large and small BECs express TMEM16A and exhibit Ca(2+)-activated Cl(-) efflux in response to extracellular nucleotides. Incubation of polarized BEC monolayers with IL-4 increased TMEM16A protein expression, membrane localization, and transepithelial secretion (I(sc)). These studies represent the first molecular identification of an alternate, noncystic fibrosis transmembrane conductance regulator, Cl(-) channel in BECs and suggest that TMEM16A may be a potential target to modulate bile formation in the treatment of cholestatic liver disorders.
Asunto(s)
Sistema Biliar/citología , Espacio Extracelular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Nucleótidos/metabolismo , Animales , Anoctamina-1 , Bilis/metabolismo , Sistema Biliar/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Canales de Cloruro , Cloro/metabolismo , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Espacio Extracelular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-4/farmacología , Proteínas de la Membrana/genética , Ratones , Proteínas de Neoplasias/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , RatasRESUMEN
The androgen receptor (AR) plays a central role in prostate cancer. Development of castration resistant prostate cancer (CRPC) requires androgen-independent activation of AR, which involves its large N-terminal domain (NTD) and entails extensive epigenetic changes depending in part on histone lysine demethylases (KDMs) that interact with AR. The AR-NTD is rich in low-complexity sequences, including a polyQ repeat. Longer polyQ sequences were reported to decrease transcriptional activity and to protect against prostate cancer, although they can lead to muscular atrophy. However, the molecular mechanisms underlying these observations are unclear. Using NMR spectroscopy, here we identify weak interactions between the AR-NTD and the KDM4A catalytic domain, and between the AR ligand-binding domain and a central KDM4A region that also contains low-complexity sequences. We also show that the AR-NTD can undergo liquid-liquid phase separation in vitro, with longer polyQ sequences phase separating more readily. Moreover, longer polyQ sequences hinder nuclear localization in the absence of hormone and increase the propensity for formation of AR-containing puncta in the nucleus of cells treated with dihydrotestosterone. These results lead us to hypothesize that polyQ-dependent liquid-liquid phase separation may provide a mechanism to decrease the transcriptional activity of AR, potentially opening new opportunities to design effective therapies against CRPC and muscular atrophy.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/genética , Péptidos/metabolismo , Receptores Androgénicos/genética , Andrógenos , Línea Celular Tumoral , Núcleo Celular/metabolismo , Expresión Génica/genética , Glutamina/metabolismo , Humanos , Masculino , Péptidos/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata Resistentes a la Castración/genética , Dominios Proteicos/genética , Receptores Androgénicos/metabolismo , Transducción de Señal/genética , Activación Transcripcional/genéticaRESUMEN
UNLABELLED: Adenosine triphosphate (ATP) is released from cholangiocytes into bile and is a potent secretogogue by increasing intracellular Ca²(+) and stimulating fluid and electrolyte secretion via binding purinergic (P2) receptors on the apical membrane. Although morphological differences exist between small and large cholangiocytes (lining small and large bile ducts, respectively), the role of P2 signaling has not been previously evaluated along the intrahepatic biliary epithelium. The aim of these studies therefore was to characterize ATP release and P2-signaling pathways in small (MSC) and large (MLC) mouse cholangiocytes. The findings reveal that both MSCs and MLCs express P2 receptors, including P2X4 and P2Y2. Exposure to extracellular nucleotides (ATP, uridine triphosphate, or 2',3'-O-[4-benzoyl-benzoyl]-ATP) caused a rapid increase in intracellular Ca²(+) concentration and in transepithelial secretion (I(sc)) in both cell types, which was inhibited by the Cl(-) channel blockers 5-nitro-2-(-3-phenylpropylamino)-benzoic acid (NPPB) or niflumic acid. In response to mechanical stimulation (flow/shear or cell swelling secondary to hypotonic exposure), both MSCs and MLCs exhibited a significant increase in the rate of exocytosis, which was paralleled by an increase in ATP release. Mechanosensitive ATP release was two-fold greater in MSCs compared to MLCs. ATP release was significantly inhibited by disruption of vesicular trafficking by monensin in both cell types. CONCLUSION: These findings suggest the existence of a P2 signaling axis along intrahepatic biliary ducts with the "upstream" MSCs releasing ATP, which can serve as a paracrine signaling molecule to "downstream" MLCs stimulating Ca²(+)-dependent secretion. Additionally, in MSCs, which do not express the cystic fibrosis transmembrane conductance regulator, Ca²(+)-activated Cl(-) efflux in response to extracellular nucleotides represents the first secretory pathway clearly identified in these cholangiocytes derived from the small intrahepatic ducts.
Asunto(s)
Adenosina Trifosfato/metabolismo , Receptores Purinérgicos P2/fisiología , Animales , Antígenos Transformadores de Poliomavirus/genética , Calcio/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Exocitosis , Ratones , Ratones Endogámicos BALB C , ARN/aislamiento & purificación , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X/genética , Receptores Purinérgicos P2X/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , TransfecciónRESUMEN
In the liver, adenosine triphosphate (ATP) is an extracellular signaling molecule that is released into bile and stimulates a biliary epithelial cell secretory response via engagement of apical P2 receptors. The molecular identities of the ion channels involved in ATP-mediated secretory responses have not been fully identified. Intermediate-conductance Ca(2+)-activated K(+) channels (IK) have been identified in biliary epithelium, but functional data are lacking. The aim of these studies therefore was to determine the location, function, and regulation of IK channels in biliary epithelial cells and to determine their potential contribution to ATP-stimulated secretion. Expression of IK-1 mRNA was found in both human Mz-Cha-1 biliary cells and polarized normal rat cholangiocyte (NRC) monolayers, and immunostaining revealed membrane localization with a predominant basolateral signal. In single Mz-Cha-1 cells, exposure to ATP activated K(+) currents, increasing current density from 1.6 +/- 0.1 to 7.6 +/- 0.8 pA/pF. Currents were dependent on intracellular Ca(2+) and sensitive to clotrimazole and TRAM-34 (specific IK channel inhibitors). Single-channel recording demonstrated that clotrimazole-sensitive K(+) currents had a unitary conductance of 46.2 +/- 1.5 pS, consistent with IK channels. In separate studies, 1-EBIO (an IK activator) stimulated K(+) currents in single cells that were inhibited by clotrimazole. In polarized NRC monolayers, ATP significantly increased transepithelial secretion which was inhibited by clotrimazole. Lastly, ATP-stimulated K(+) currents were inhibited by the P2Y receptor antagonist suramin and by the inositol 1,4,5-triphosphate (IP3) receptor inhibitor 2-APB. Together these studies demonstrate that IK channels are present in biliary epithelial cells and contribute to ATP-stimulated secretion through a P2Y-IP3 receptor pathway.
Asunto(s)
Sistema Biliar/fisiología , Células Epiteliales/fisiología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/fisiología , Adenosina Trifosfato/farmacología , Animales , Apamina/farmacología , Bario/farmacología , Bencimidazoles/farmacología , Sistema Biliar/citología , Tampones (Química) , Línea Celular Tumoral , Membrana Celular/metabolismo , Células Cultivadas , Quelantes/farmacología , Clotrimazol/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Fenómenos Electrofisiológicos , Células Epiteliales/efectos de los fármacos , Expresión Génica/genética , Humanos , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inhibidores , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/agonistas , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Modelos Biológicos , Técnicas de Placa-Clamp , Antagonistas del Receptor Purinérgico P2 , Pirazoles/farmacología , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Suramina/farmacologíaRESUMEN
5'-AMP-activated kinase (AMPK) plays a key role in the regulation of cellular lipid metabolism. The contribution of vesicular exocytosis to this regulation is not known. Accordingly, we studied the effects of AMPK on exocytosis and intracellular lipid content in a model liver cell line. Activation of AMPK by metformin or 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) increased the rates of constitutive exocytosis by about 2-fold. Stimulation of exocytosis by AMPK occurred within minutes, and persisted after overnight exposure to metformin or AICAR. Activation of AMPK also increased the amount of triacylglycerol (TG) and apolipoprotein B (apoB) secreted from lipid-loaded cells. These effects were accompanied by a decrease in the intracellular lipid content indicating that exocytosis of lipoproteins was involved in these lipid-lowering effects. While AMPK increased the rates of fatty acid oxidation (FAO), the lipid-lowering effects were quantitatively significant even after inhibition of FAO with R-etomoxir. These results suggest that hepatic AMPK stimulates constitutive exocytosis of lipoproteins, which may function in parallel with FAO to regulate intracellular lipid content.
Asunto(s)
Apolipoproteínas B/metabolismo , Exocitosis/fisiología , Hepatocitos/metabolismo , Complejos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Triglicéridos/metabolismo , Proteínas Quinasas Activadas por AMP , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Amiodarona/farmacología , Animales , Línea Celular , Inhibidores Enzimáticos/farmacología , Compuestos Epoxi/farmacología , Exocitosis/efectos de los fármacos , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Hepatocitos/citología , Humanos , Hipoglucemiantes/farmacología , Metabolismo de los Lípidos , Lovastatina/análogos & derivados , Lovastatina/metabolismo , Metformina/farmacología , Complejos Multienzimáticos/genética , Oxidación-Reducción , Técnicas de Placa-Clamp , Proteínas Serina-Treonina Quinasas/genética , Ratas , Ribonucleótidos/farmacología , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Triglicéridos/químicaRESUMEN
This protocol describes reconstitution assays to study how the neurotransmitter release machinery triggers Ca2+-dependent synaptic vesicle fusion. The assays monitor fusion between proteoliposomes containing the synaptic vesicle SNARE synaptobrevin (with or without the Ca2+ sensor synaptotagmin-1) and proteoliposomes initially containing the plasma membrane SNAREs syntaxin-1 and soluble NSF attachment protein (SNAP)-25. Lipid mixing (from fluorescence de-quenching of Marina-Blue-labeled lipids) and content mixing (from development of fluorescence resonance energy transfer (FRET) between phycoerythrin-biotin (PhycoE-Biotin) and Cy5-streptavidin trapped in the two proteoliposome populations) are measured simultaneously to ensure that true, nonleaky membrane fusion is monitored. This protocol is based on a method developed to study yeast vacuolar fusion. In contrast to other protocols used to study the release machinery, this assay incorporates N-ethylmaleimide sensitive factor (NSF) and α-SNAP, which disassemble syntaxin-1 and SNAP-25 heterodimers. As a result, fusion requires Munc18-1, which binds to the released syntaxin-1, and Munc13-1, which, together with Munc18-1, orchestrates SNARE complex assembly. The protocol can be readily adapted to investigation of other types of intracellular membrane fusion by using appropriate alternative proteins. Total time required for one round of the assay is 4 d.
Asunto(s)
Fusión de Membrana/fisiología , Modelos Biológicos , Proteínas SNARE/química , Proteínas SNARE/metabolismo , Vesículas Sinápticas/química , Vesículas Sinápticas/metabolismo , Colorantes/química , Colorantes/metabolismo , Lípidos/química , Liposomas/química , Liposomas/metabolismo , Transmisión Sináptica , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Munc13-1 acts as a master regulator of neurotransmitter release, mediating docking-priming of synaptic vesicles and diverse presynaptic plasticity processes. It is unclear how the functions of the multiple domains of Munc13-1 are coordinated. The crystal structure of a Munc13-1 fragment including its C1, C2B and MUN domains (C1C2BMUN) reveals a 19.5 nm-long multi-helical structure with the C1 and C2B domains packed at one end. The similar orientations of the respective diacyglycerol- and Ca2+-binding sites of the C1 and C2B domains suggest that the two domains cooperate in plasma-membrane binding and that activation of Munc13-1 by Ca2+ and diacylglycerol during short-term presynaptic plasticity are closely interrelated. Electrophysiological experiments in mouse neurons support the functional importance of the domain interfaces observed in C1C2BMUN. The structure imposes key constraints for models of neurotransmitter release and suggests that Munc13-1 bridges the vesicle and plasma membranes from the periphery of the membrane-membrane interface.
Asunto(s)
Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Neurotransmisores/metabolismo , Animales , Células Cultivadas , Cristalografía por Rayos X , Ratones , Modelos Moleculares , Neuronas/fisiología , Conformación Proteica , RatasRESUMEN
Neurotransmitter release requires SNARE complexes to bring membranes together, NSF-SNAPs to recycle the SNAREs, Munc18-1 and Munc13s to orchestrate SNARE complex assembly, and Synaptotagmin-1 to trigger fast Ca(2+)-dependent membrane fusion. However, it is unclear whether Munc13s function upstream and/or downstream of SNARE complex assembly, and how the actions of their multiple domains are integrated. Reconstitution, liposome-clustering and electrophysiological experiments now reveal a functional synergy between the C1, C2B and C2C domains of Munc13-1, indicating that these domains help bridging the vesicle and plasma membranes to facilitate stimulation of SNARE complex assembly by the Munc13-1 MUN domain. Our reconstitution data also suggest that Munc18-1, Munc13-1, NSF, αSNAP and the SNAREs are critical to form a 'primed' state that does not fuse but is ready for fast fusion upon Ca(2+) influx. Overall, our results support a model whereby the multiple domains of Munc13s cooperate to coordinate synaptic vesicle docking, priming and fusion.
Asunto(s)
Membrana Celular/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Dominios Proteicos , Multimerización de Proteína , Ratas , Proteínas SNARE/metabolismoRESUMEN
Lipid perturbations associated with triglyceride overstorage in beta-cells impair insulin secretion, a process termed lipotoxicity. To assess the role of hormone-sensitive lipase, which is expressed and enzymatically active in beta-cells, in the development of lipotoxicity, we generated transgenic mice overexpressing hormone-sensitive lipase specifically in beta-cells. Transgenic mice developed glucose intolerance and severely blunted glucose-stimulated insulin secretion when challenged with a high-fat diet. As expected, both lipase activity and forskolin-stimulated lipolysis was increased in transgenic compared with wild-type islets. This was reflected in significantly lower triglycerides levels in transgenic compared with wild-type islets in mice receiving the high-fat diet, whereas no difference in islet triglycerides was found between the two genotypes under low-fat diet conditions. Our results highlight the importance of mobilization of the islet triglyceride pool in the development of beta-cell lipotoxicity. We propose that hormone-sensitive lipase is involved in mediating beta-cell lipotoxicity by providing ligands for peroxisome proliferator-activated receptors and other lipid-activated transcription factors, which in turn alter the expression of critical genes. One such gene might be uncoupling protein-2, which was found to be upregulated in transgenic islets, a change that was accompanied by decreased ATP levels.
Asunto(s)
Islotes Pancreáticos/enzimología , Proteínas de Transporte de Membrana , Proteínas Mitocondriales , Esterol Esterasa/genética , Esterol Esterasa/metabolismo , Triglicéridos/metabolismo , Animales , Glucemia/metabolismo , Peso Corporal , Ácidos Grasos/metabolismo , Femenino , Regulación Enzimológica de la Expresión Génica , Transportador de Glucosa de Tipo 2 , Inmunohistoquímica , Canales Iónicos , Islotes Pancreáticos/química , Islotes Pancreáticos/crecimiento & desarrollo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Proteínas de Transporte de Monosacáridos/análisis , Oxidación-Reducción , Fenotipo , Proteínas/metabolismo , Ratas , Proteína Desacopladora 2 , Regulación hacia ArribaRESUMEN
Genetic predisposition and environmental influences insidiously converge to cause glucose intolerance and hyperglycemia. Beta-cell compensates by secreting more insulin and when it fails, overt diabetes mellitus ensues. The need to understand the mechanisms involved in insulin secretion cannot be stressed enough. Phosphorylation of proteins plays an important role in regulating insulin secretion. In order to understand how a particular cellular process is regulated by protein phosphorylation the nature of the protein kinases and protein phosphatases involved and the mechanisms that determine when and where these enzymes are active should be investigated. While the actions of protein kinases have been intensely studied within the beta-cell, less emphasis has been placed on protein phosphatases even though they play an important regulatory role. This review focuses on the importance of protein phosphatase 2A in insulin secretion. Most of the present knowledge on protein phosphatase 2A originates from protein phosphatase inhibitor studies on islets and beta-cell lines. The ability of protein phosphatase 2A to change its activity in the presence of glucose and inhibitors provides clues to its role in regulating insulin secretion. An aggressive approach to elucidate the substrates and mechanisms of action of protein phosphatases is crucial to the understanding of phosphorylation events within the beta-cell. Characterizing protein phosphatase 2A within the beta-cell will certainly help us in understanding the mechanisms involved in insulin secretion and provide valuable information for drug development.
Asunto(s)
Diabetes Mellitus Tipo 2/enzimología , Insulina/metabolismo , Islotes Pancreáticos/enzimología , Fosfoproteínas Fosfatasas/fisiología , Calcineurina/fisiología , Humanos , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Proteína Fosfatasa 2RESUMEN
OBJECTIVE: To validate magnetic resonance spectroscopy (MRS) as a tool for non-invasive quantification of pancreatic triglyceride (TG) content and to measure the pancreatic TG content in a diverse human population with a wide range of body mass index (BMI) and glucose control. METHODS: To validate the MRS method, we measured TG content in the pancreatic tissue of 12 lean and 12 fatty ZDF rats (ages 5-14 weeks) both by MRS and the gold standard biochemical assay. We used MRS to measure pancreatic TG content in vivo in 79 human volunteers. Additionally, to assess the reproducibility of the method, in 33 volunteers we obtained duplicate MRS measurements 1-2 weeks apart. RESULTS: MRS quantifies pancreatic TG content with high reproducibility and concordance to the biochemical measurement (Spearman's rank correlation coefficient = 0.91). In humans, median pancreatic TG content was as follows: (1) normal weight and normoglycemic group 0.46 f/w%, (2) overweight or obese but normoglycemic group 3.16 f/w%, (3) impaired fasting glucose or impaired glucose tolerance group (BMI matched with group 2) 5.64 f/w%, and (4) untreated type 2 diabetes group (BMI matched with group 2) 5.54 f/w% (Jonckheere-Terpstra trend test across groups p < 0.001). CONCLUSIONS: Human pancreatic steatosis, as measured by MRS, increases with BMI and with impaired glycemia. MRS is a quantitative and reproducible non-invasive clinical research tool which will enable systematic studies of the relationship between ectopic fat accumulation in the pancreas and development of type 2 diabetes.
Asunto(s)
Espectroscopía de Resonancia Magnética , Páncreas/metabolismo , Triglicéridos/metabolismo , Adulto , Análisis de Varianza , Animales , Índice de Masa Corporal , Diabetes Mellitus Tipo 2/metabolismo , Femenino , Prueba de Tolerancia a la Glucosa , Humanos , Masculino , Análisis Multivariante , Páncreas/patología , Ratas , Ratas Zucker , Reproducibilidad de los ResultadosRESUMEN
Mutations in 1-acylglycerol-3-phosphate-O-acyltransferase 2 (AGPAT2) cause congenital generalized lipodystrophy. To understand the molecular mechanisms underlying the metabolic complications associated with AGPAT2 deficiency, Agpat2 null mice were generated. Agpat2(-/-) mice develop severe lipodystrophy affecting both white and brown adipose tissue, extreme insulin resistance, diabetes, and hepatic steatosis. The expression of lipogenic genes and rates of de novo fatty acid biosynthesis were increased approximately 4-fold in Agpat2(-/-) mouse livers. The mRNA and protein levels of monoacylglycerol acyltransferase isoform 1 were markedly increased in the livers of Agpat2(-/-) mice, suggesting that the alternative monoacylglycerol pathway for triglyceride biosynthesis is activated in the absence of AGPAT2. Feeding a fat-free diet reduced liver triglycerides by approximately 50% in Agpat2(-/-) mice. These observations suggest that both dietary fat and hepatic triglyceride biosynthesis via a monoacylglycerol pathway may contribute to hepatic steatosis in Agpat2(-/-) mice.
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1-Acilglicerol-3-Fosfato O-Aciltransferasa/metabolismo , Hígado Graso/metabolismo , Resistencia a la Insulina/genética , Lipodistrofia Generalizada Congénita/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferasa/genética , Tejido Adiposo/metabolismo , Animales , Metabolismo Energético , Lipodistrofia Generalizada Congénita/genética , Ratones , Ratones Noqueados , Modelos Animales , Triglicéridos/biosíntesisRESUMEN
OBJECTIVE: Fluxes through mitochondrial pathways are defective in insulin-resistant skeletal muscle, but it is unclear whether similar mitochondrial defects play a role in the liver during insulin resistance and/or diabetes. The purpose of this study is to determine whether abnormal mitochondrial metabolism plays a role in the dysregulation of both hepatic fat and glucose metabolism during diabetes. RESEARCH DESIGN AND METHODS: Mitochondrial fluxes were measured using (2)H/(13)C tracers and nuclear magnetic resonance spectroscopy in ZDF rats during early and advanced diabetes. To determine whether defects in hepatic fat oxidation can be corrected by peroxisome proliferator-activated receptor (PPAR-)-alpha activation, rats were treated with WY14,643 for 3 weeks before tracer administration. RESULTS: Hepatic mitochondrial fat oxidation in the diabetic liver was impaired twofold secondary to decreased ketogenesis, but tricarboxylic acid (TCA) cycle activity and pyruvate carboxylase flux were normal in newly diabetic rats and elevated in older rats. Treatment of diabetic rats with a PPAR-alpha agonist induced hepatic fat oxidation via ketogenesis and hepatic TCA cycle activity but failed to lower fasting glycemia or endogenous glucose production. In fact, PPAR-alpha agonism overstimulated mitochondrial TCA cycle flux and induced pyruvate carboxylase flux and gluconeogenesis in lean rats. CONCLUSIONS: The impairment of certain mitochondrial fluxes, but preservation or induction of others, suggests a complex defect in mitochondrial metabolism in the diabetic liver. These data indicate an important codependence between hepatic fat oxidation and gluconeogenesis in the normal and diabetic state and potentially explain the sometimes equivocal effect of PPAR-alpha agonists on glycemia.
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Hígado/metabolismo , Mitocondrias/metabolismo , PPAR alfa/agonistas , Pirimidinas/farmacología , Animales , Glucemia/metabolismo , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatología , Ácidos Grasos no Esterificados/sangre , Gluconeogénesis , Glucosa/metabolismo , Insulina/sangre , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Mitocondrias/efectos de los fármacos , Radioinmunoensayo , Ratas , Ratas Zucker , Triglicéridos/sangreRESUMEN
Peroxisome proliferator-activated receptor alpha (PPARalpha) regulates the utilization of fat as an energy source during starvation and is the molecular target for the fibrate dyslipidemia drugs. Here, we identify the endocrine hormone fibroblast growth factor 21 (FGF21) as a mediator of the pleiotropic actions of PPARalpha. FGF21 is induced directly by PPARalpha in liver in response to fasting and PPARalpha agonists. FGF21 in turn stimulates lipolysis in white adipose tissue and ketogenesis in liver. FGF21 also reduces physical activity and promotes torpor, a short-term hibernation-like state of regulated hypothermia that conserves energy. These findings demonstrate an unexpected role for the PPARalpha-FGF21 endocrine signaling pathway in regulating diverse metabolic and behavioral aspects of the adaptive response to starvation.
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Ayuno/fisiología , Factores de Crecimiento de Fibroblastos/fisiología , Hepatocitos/metabolismo , Hígado/metabolismo , PPAR alfa/metabolismo , Adenoviridae/genética , Tejido Adiposo/metabolismo , Animales , Células Cultivadas , Inmunoprecipitación de Cromatina , Femenino , Humanos , Immunoblotting , Lipólisis/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Plásmidos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiología , TransfecciónRESUMEN
Carnitine palmitoyltransferase II (CPT-II) has a crucial role in the beta-oxidation of long-chain fatty acids in mitochondria. We report here the crystal structure of rat CPT-II at 1.9A resolution. The overall structure shares strong similarity to those of short- and medium-chain carnitine acyltransferases, although detailed structural differences in the active site region have a significant impact on the substrate selectivity of CPT-II. Three aliphatic chains, possibly from a detergent that is used for the crystallization, were found in the structure. Two of them are located in the carnitine and CoA binding sites, respectively. The third aliphatic chain may mimic the long-chain acyl group in the substrate of CPT-II. The binding site for this aliphatic chain does not exist in the short- and medium-chain carnitine acyltransferases, due to conformational differences among the enzymes. A unique insert in CPT-II is positioned on the surface of the enzyme, with a highly hydrophobic surface. It is likely that this surface patch mediates the association of CPT-II with the inner membrane of the mitochondria.