RESUMO
Sirtuins are a family of proteins that regulate biological processes such as cellular stress and aging by removing posttranslational modifications (PTMs). We recently identified several novel PTMs that can be removed by sirtuin 4 (SIRT4), which is found in mitochondria. We showed that mice with a global loss of SIRT4 [SIRT4-knockout (KO) mice] developed an increase in glucose- and leucine-stimulated insulin secretion, and this was followed by accelerated age-induced glucose intolerance and insulin resistance. Because whole body SIRT4-KO mice had alterations to nutrient-stimulated insulin secretion, we hypothesized that SIRT4 plays a direct role in regulating pancreatic ß-cell function. Thus, we tested whether ß-cell-specific ablation of SIRT4 would recapitulate the elevated insulin secretion seen in mice with a global loss of SIRT4. Tamoxifen-inducible ß-cell-specific SIRT4-KO mice were generated, and their glucose tolerance and glucose- and leucine-stimulated insulin secretion were measured over time. These mice exhibited normal glucose- and leucine-stimulated insulin secretion and maintained normal glucose tolerance even as they aged. Furthermore, 832/13 ß-cells with a CRISPR/Cas9n-mediated loss of SIRT4 did not show any alterations in nutrient-stimulated insulin secretion. Despite the fact that whole body SIRT4-KO mice demonstrated an age-induced increase in glucose- and leucine-stimulated insulin secretion, our current data indicate that the loss of SIRT4 specifically in pancreatic ß-cells, both in vivo and in vitro, does not have a significant impact on nutrient-stimulated insulin secretion. These data suggest that SIRT4 controls nutrient-stimulated insulin secretion during aging by acting on tissues external to the ß-cell, which warrants further study.
Assuntos
Secreção de Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Proteínas Mitocondriais/metabolismo , Sirtuínas/metabolismo , Animais , Glucose/farmacologia , Intolerância à Glucose/metabolismo , Resistência à Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Leucina/farmacologia , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Nutrientes , Processamento de Proteína Pós-TraducionalRESUMO
After multiple decades of investigation, the precise mechanisms involved in fuel-stimulated insulin secretion are still being revealed. One avenue for gaining deeper knowledge is to apply emergent tools of "metabolomics," involving mass spectrometry and nuclear magnetic resonance-based profiling of islet cells in their fuel-stimulated compared with basal states. The current article summarizes recent insights gained from application of metabolomics tools to the specific process of glucose-stimulated insulin secretion, revealing 2 new mechanisms that may provide targets for improving insulin secretion in diabetes.
Assuntos
Pesquisa Biomédica/métodos , Ilhotas Pancreáticas/metabolismo , Metabolômica/métodos , Modelos Biológicos , Animais , Pesquisa Biomédica/tendências , Exocitose , Glucose/metabolismo , Humanos , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/enzimologia , Metabolômica/tendências , Via SecretóriaRESUMO
SIRT3 is a nicotinamide adenine dinucleotide (NAD+)-dependent mitochondrial protein deacetylase purported to influence metabolism through post-translational modification of metabolic enzymes. Fuel-stimulated insulin secretion, which involves mitochondrial metabolism, could be susceptible to SIRT3-mediated effects. We used CRISPR/Cas9 technology to manipulate SIRT3 expression in ß cells, resulting in widespread SIRT3-dependent changes in acetylation of key metabolic enzymes but no appreciable changes in glucose- or pyruvate-stimulated insulin secretion or metabolomic profile during glucose stimulation. Moreover, these broad changes in the SIRT3-targeted acetylproteome did not affect responses to nutritional or ER stress. We also studied mice with global SIRT3 knockout fed either standard chow (STD) or high-fat and high-sucrose (HFHS) diets. Only when chronically fed HFHS diet do SIRT3 KO animals exhibit a modest reduction in insulin secretion. We conclude that broad changes in mitochondrial protein acetylation in response to manipulation of SIRT3 are not sufficient to cause changes in islet function or metabolism.
Assuntos
Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Hipernutrição/metabolismo , Proteoma/metabolismo , Sirtuína 3/metabolismo , Acetilação , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Células Clonais , Dieta Hiperlipídica , Glucose/farmacologia , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , Metaboloma , Camundongos Knockout , Proteínas Mitocondriais/metabolismo , Mutação/genética , Ratos , Secretagogos/metabolismo , Sacarose , TransgenesRESUMO
Increased ß-cell death coupled with the inability to replicate existing ß-cells drives the decline in ß-cell mass observed in the progression of both major forms of diabetes. Understanding endogenous mechanisms of islet cell survival could have considerable value for the development of novel strategies to limit ß-cell loss and thereby promote ß-cell recovery. Insulinoma cells have provided useful insight into ß-cell death pathways but observations made in cell lines sometimes fail to translate to primary islets. Here, we report dramatic differences in the temporal regulation and engagement of the apoptotic program in primary rodent islets relative to the INS-1 derived 832/13 cell line. As expected, 832/13 cells rapidly induced cell stress markers in response to ER stress or DNA damage and were fully committed to apoptosis, resulting in >80% cell death within 24 h. In contrast, primary rat islets were largely refractory to cell death in response to ER stress and DNA damage, despite rapid induction of stress markers, such as XBP-1(s), CHOP, and PUMA. Gene expression profiling revealed a general suppression of pro-apoptotic machinery, such as Apaf-1 and caspase 3, and sustained levels of pro-survival factors, such as cIAP-1, cIAP-2, and XIAP, in rat islets. Furthermore, we observed sustained induction of autophagy following chronic ER stress and found that inhibition of autophagy rendered islet ß-cells highly vulnerable to ER stress-induced cell death. We propose that islet ß-cells dampen the apoptotic response to delay the onset of cell death, providing a temporal window in which autophagy can be activated to limit cellular damage and promote survival.
Assuntos
Apoptose/fisiologia , Autofagia/fisiologia , Ilhotas Pancreáticas/citologia , Animais , Fator Apoptótico 1 Ativador de Proteases , Caspase 3/metabolismo , Linhagem Celular , Sobrevivência Celular/fisiologia , Células Cultivadas , Estresse do Retículo Endoplasmático , Glucose/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Insulina/metabolismo , Secreção de Insulina , Insulinoma/patologia , Ilhotas Pancreáticas/fisiologia , Neoplasias Pancreáticas/patologia , RatosRESUMO
Sirtuins are NAD+-dependent protein deacylases that regulate several aspects of metabolism and aging. In contrast to the other mammalian sirtuins, the primary enzymatic activity of mitochondrial sirtuin 4 (SIRT4) and its overall role in metabolic control have remained enigmatic. Using a combination of phylogenetics, structural biology, and enzymology, we show that SIRT4 removes three acyl moieties from lysine residues: methylglutaryl (MG)-, hydroxymethylglutaryl (HMG)-, and 3-methylglutaconyl (MGc)-lysine. The metabolites leading to these post-translational modifications are intermediates in leucine oxidation, and we show a primary role for SIRT4 in controlling this pathway in mice. Furthermore, we find that dysregulated leucine metabolism in SIRT4KO mice leads to elevated basal and stimulated insulin secretion, which progressively develops into glucose intolerance and insulin resistance. These findings identify a robust enzymatic activity for SIRT4, uncover a mechanism controlling branched-chain amino acid flux, and position SIRT4 as a crucial player maintaining insulin secretion and glucose homeostasis during aging.
Assuntos
Amidoidrolases/metabolismo , Insulina/metabolismo , Leucina/metabolismo , Lisina/metabolismo , Proteínas Mitocondriais/metabolismo , Sirtuínas/metabolismo , Sequência de Aminoácidos , Animais , Carbono-Carbono Ligases/metabolismo , Glucose/metabolismo , Células HEK293 , Homeostase , Humanos , Resistência à Insulina , Secreção de Insulina , Análise do Fluxo Metabólico , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mitocondriais/química , Modelos Moleculares , Filogenia , Sirtuínas/químicaRESUMO
The mitochondrial sirtuin SIRT3 is a protein deacylase that influences almost every major aspect of mitochondrial biology, including nutrient oxidation, ATP generation, reactive oxygen species (ROS) detoxification, mitochondrial dynamics, and the mitochondrial unfolded protein response (UPR). Interestingly, mice lacking SIRT3 (SIRT3KO), either spontaneously or when crossed with mouse models of disease, develop several diseases of aging at an accelerated pace, such as cancer, metabolic syndrome, cardiovascular disease, and neurodegenerative diseases, and, thus, might be a valuable model of accelerated aging. In this review, we discuss functions of SIRT3 in pathways involved in diseases of aging and how the lack of SIRT3 might accelerate the aging process. We also suggest that further studies on SIRT3 will help uncover important new pathways driving the aging process.
Assuntos
Envelhecimento/genética , Doença/genética , Sirtuína 3/genética , Animais , Humanos , Longevidade , CamundongosRESUMO
Insulin secretion from ß cells of the pancreatic islets of Langerhans controls metabolic homeostasis and is impaired in individuals with type 2 diabetes (T2D). Increases in blood glucose trigger insulin release by closing ATP-sensitive K+ channels, depolarizing ß cells, and opening voltage-dependent Ca2+ channels to elicit insulin exocytosis. However, one or more additional pathway(s) amplify the secretory response, likely at the distal exocytotic site. The mitochondrial export of isocitrate and engagement with cytosolic isocitrate dehydrogenase (ICDc) may be one key pathway, but the mechanism linking this to insulin secretion and its role in T2D have not been defined. Here, we show that the ICDc-dependent generation of NADPH and subsequent glutathione (GSH) reduction contribute to the amplification of insulin exocytosis via sentrin/SUMO-specific protease-1 (SENP1). In human T2D and an in vitro model of human islet dysfunction, the glucose-dependent amplification of exocytosis was impaired and could be rescued by introduction of signaling intermediates from this pathway. Moreover, islet-specific Senp1 deletion in mice caused impaired glucose tolerance by reducing the amplification of insulin exocytosis. Together, our results identify a pathway that links glucose metabolism to the amplification of insulin secretion and demonstrate that restoration of this axis rescues ß cell function in T2D.
Assuntos
Diabetes Mellitus Tipo 2/fisiopatologia , Endopeptidases/fisiologia , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Isocitratos/metabolismo , Animais , Domínio Catalítico , Membrana Celular/metabolismo , Cisteína Endopeptidases , Diabetes Mellitus Tipo 2/patologia , Endopeptidases/biossíntese , Endopeptidases/deficiência , Endopeptidases/genética , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Técnicas de Inativação de Genes , Glucose/metabolismo , Glucose/farmacologia , Glutationa/farmacologia , Células HEK293 , Homeostase , Humanos , Insulina/farmacologia , Secreção de Insulina , Ilhotas Pancreáticas/fisiopatologia , Isocitrato Desidrogenase/fisiologia , Isocitratos/farmacologia , Masculino , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos C57BL , NADP/metabolismo , Especificidade de Órgãos , Interferência de RNA , Proteínas Recombinantes de Fusão/metabolismo , Vesículas Secretórias/metabolismo , Transdução de Sinais , SumoilaçãoRESUMO
We report the identification and characterization of a five-carbon protein posttranslational modification (PTM) called lysine glutarylation (Kglu). This protein modification was detected by immunoblot and mass spectrometry (MS), and then comprehensively validated by chemical and biochemical methods. We demonstrated that the previously annotated deacetylase, sirtuin 5 (SIRT5), is a lysine deglutarylase. Proteome-wide analysis identified 683 Kglu sites in 191 proteins and showed that Kglu is highly enriched on metabolic enzymes and mitochondrial proteins. We validated carbamoyl phosphate synthase 1 (CPS1), the rate-limiting enzyme in urea cycle, as a glutarylated protein and demonstrated that CPS1 is targeted by SIRT5 for deglutarylation. We further showed that glutarylation suppresses CPS1 enzymatic activity in cell lines, mice, and a model of glutaric acidemia type I disease, the last of which has elevated glutaric acid and glutaryl-CoA. This study expands the landscape of lysine acyl modifications and increases our understanding of the deacylase SIRT5.