RESUMO
AIMS/HYPOTHESIS: Sirtuin 1 (SIRT1) has emerged as a key metabolic regulator of glucose homeostasis and insulin secretion. Enhanced SIRT1 activity has been shown to be protective against diabetes, although the mechanisms remain largely unknown. The aim of this study was to determine how SIRT1 regulates insulin secretion in the pancreatic beta cell. METHODS: Pancreatic beta cell-specific Sirt1 deletion was induced by tamoxifen injection in 9-week-old Pdx1CreER:floxSirt1 mice (Sirt1BKO). Controls were injected with vehicle. Mice were assessed metabolically via glucose challenge, insulin tolerance tests and physical variables. In parallel, Sirt1 short interfering RNA-treated MIN6 cells (SIRT1KD) and isolated Sirt1BKO islets were used to investigate the effect of SIRT1 inactivation on insulin secretion and gene expression. RESULTS: OGTTs showed impaired glucose disposal in Sirt1BKO mice due to insufficient insulin secretion. Isolated Sirt1BKO islets and SIRT1KD MIN6 cells also exhibited impaired glucose-stimulated insulin secretion. Subsequent analyses revealed impaired α-ketoisocaproic acid-induced insulin secretion and attenuated glucose-induced Ca(2+) influx, but normal insulin granule exocytosis in Sirt1BKO beta cells. Microarray studies revealed a large cluster of mitochondria-related genes, the expression of which was dysregulated in SIRT1KD MIN6 cells. Upon further analysis, we demonstrated an explicit defect in mitochondrial function: the inability to couple nutrient metabolism to mitochondrial membrane hyperpolarisation and reduced oxygen consumption rates. CONCLUSIONS/INTERPRETATION: Taken together, these findings indicate that in beta cells the deacetylase SIRT1 regulates the expression of specific mitochondria-related genes that control metabolic coupling, and that a decrease in beta cell Sirt1 expression impairs glucose sensing and insulin secretion.
Assuntos
Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Sirtuína 1/metabolismo , Animais , Western Blotting , Feminino , Imuno-Histoquímica , Masculino , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase em Tempo Real , Sirtuína 1/genéticaRESUMO
Genomewide association studies have linked a polymorphism in the zinc transporter 8 (Znt8) gene to higher risk of developing type 2 diabetes. Znt8 is highly expressed in pancreatic ß-cells where it is involved in the regulation of zinc transport into granules. However, Znt8 is also expressed in other tissues including α-cells, where its function is as yet unknown. Previous work demonstrated that mice lacking Znt8 globally were more susceptible to diet-induced obesity (Lemaire et al., Proc Natl Acad Sci USA 106: 14872-14877, 2009; Nicolson et al., Diabetes 58: 2070-2083, 2009). Therefore, the main goal of this study was to examine the physiological impact of ß-cell-specific Znt8 deficiency in mice during high-fat high-calorie (HFHC) diet feeding. For these studies, we used ß-cell-specific Znt8 knockout (Ins2Cre:Znt8loxP/loxP) and whole body Znt8 knockout (Cre-:Znt8(-/-)) mice placed on a HFHC diet for 16 wk. Ins2Cre:Znt8loxP/loxP mice on HFHC diet had similar body weights throughout the study but displayed impaired insulin biosynthesis and secretion and were glucose intolerant compared with littermate control Ins2Cre mice. In contrast, Cre-:Znt8(-/-) mice became remarkably obese, hyperglycemic, hyperinsulinemic, insulin resistant, and glucose intolerant compared with littermate control Cre- mice. These data show that ß-cell Znt8 alone does not considerably aggravate weight gain and glucose intolerance during metabolic stress imposed by an HFHC diet. However, global loss of Znt8 is involved in exacerbating diet-induced obesity and resulting insulin resistance, and this may be due to the loss of Znt8 activity in a tissue other than the ß-cell. Thus, our data suggest that Znt8 contributes to the risk of developing type 2 diabetes through ß-cell- and non-ß-cell-specific effects.
Assuntos
Proteínas de Transporte de Cátions/fisiologia , Dieta Hiperlipídica , Resistência à Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Obesidade/metabolismo , Análise de Variância , Animais , Glicemia/metabolismo , Proteínas de Transporte de Cátions/deficiência , Proteínas de Transporte de Cátions/genética , Ingestão de Energia , Resistência à Insulina/genética , Masculino , Análise por Pareamento , Camundongos , Camundongos Knockout , Obesidade/genética , Estresse Fisiológico/fisiologia , Distribuição Tecidual , Transportador 8 de ZincoRESUMO
AIMS/HYPOTHESIS: Using a novel directed differentiation protocol, we recently generated up to 25% insulin-producing cells from human embryonic stem cells (hESCs) (insulin(+) cells). At this juncture, it was important to functionally and molecularly characterise these hESC-derived insulin(+) cells and identify key differences and similarities between them and primary beta cells. METHODS: We used a new reporter hESC line with green fluorescent protein (GFP) cDNA targeted to the INS locus by homologous recombination (INS (GFP/w)) and an untargeted hESC line (HES2). INS (GFP/w) allowed efficient identification and purification of GFP-producing (INS:GFP(+)) cells. Insulin(+) cells were examined for key features of adult beta cells using microarray, quantitative PCR, secretion assays, imaging and electrophysiology. RESULTS: Immunofluorescent staining showed complete co-localisation of insulin with GFP; however, cells were often multihormonal, many with granules containing insulin and glucagon. Electrophysiological recordings revealed variable K(ATP) and voltage-gated Ca(2+) channel activity, and reduced glucose-induced cytosolic Ca(2+) uptake. This translated into defective glucose-stimulated insulin secretion but, intriguingly, appropriate glucagon responses. Gene profiling revealed differences in global gene expression between INS:GFP(+) cells and adult human islets; however, INS:GFP(+) cells had remarkably similar expression of endocrine-lineage transcription factors and genes involved in glucose sensing and exocytosis. CONCLUSIONS/INTERPRETATION: INS:GFP(+) cells can be purified from differentiated hESCs, providing a superior source of insulin-producing cells. Genomic analyses revealed that INS:GFP(+) cells collectively resemble immature endocrine cells. However, insulin(+) cells were heterogeneous, a fact that translated into important functional differences within this population. The information gained from this study may now be used to generate new iterations of functioning beta cells that can be purified for transplant.
Assuntos
Células-Tronco Embrionárias/citologia , Células Secretoras de Insulina/citologia , Insulina/metabolismo , Trifosfato de Adenosina/química , Adulto , Animais , Cálcio/metabolismo , Eletrofisiologia/métodos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Ilhotas Pancreáticas/citologia , Camundongos , Microscopia de Fluorescência/métodos , Análise de Sequência com Séries de Oligonucleotídeos , Pâncreas/embriologia , Potássio/metabolismo , Fatores de TempoRESUMO
Peptide hormones encoded by the proglucagon (Gcg) and glucose-dependent insulinotropic polypeptide (Gip) genes are evolutionarily related glucagon-like sequences and act through a subfamily of G-protein-coupled receptors. A better understanding of the evolutionary history of these hormones and receptors should yield insight into their biological functions. The availability of a large number of near-complete vertebrate genome sequences is a powerful resource to address questions concerning the evolution of sequences; here, we utilize these resources to examine the evolution of glucagon-like sequences and their receptors. These studies led to the discovery of novel genes for a glucagon receptor-like receptor (Grlr) and a glucagon-like sequence (exendin) in vertebrates. Both exendin and GRLR have ancient origins, early in vertebrate evolution, but have been lost on the ancestral lineage leading to extant mammals. We also show that exendin and GRLR are both expressed in the brain of the chicken and Xenopus tropicals, results that suggest that the products of these genes function in this tissue. The lack of exendin or Grlr genes in mammals suggests that other genes may have acquired the functions of exendin and Grlr during mammalian evolution.
Assuntos
Polipeptídeo Inibidor Gástrico/genética , Peptídeos Semelhantes ao Glucagon/genética , Glucagon/genética , Incretinas/genética , Mamíferos/genética , Receptores de Glucagon/genética , Animais , Sequência de Bases/genética , Galinhas/genética , Glucagon/fisiologia , Humanos , Incretinas/fisiologia , Filogenia , Precursores de Proteínas/genética , Receptores de Glucagon/fisiologia , Xenopus/genéticaRESUMO
The allocation of processing resources during spoken discourse comprehension was studied in a manner analogous to self-paced reading using the auditory moving window technique (Ferreira, Henderson, Anes, Weeks, & McFarlane, 1996). Young and older participants listened to spoken passages in a self-paced segment-by-segment fashion. In Experiment 1, we examined the influence of speech rate and passage complexity on discourse encoding and recall performance. In Experiment 2, we examined the influence of speech rate and presentation mode (self-paced vs. full-passage presentation) on recall performance. Results suggest that diminished memory performance in the older adult group relative to the young adult group is attributable to age-related differences in how resources were allocated during the initial encoding of the spoken discourse.