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1.
Diabetologia ; 65(6): 997-1011, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35294578

RESUMEN

AIMS/HYPOTHESIS: Although targeted in extrapancreatic tissues by several drugs used to treat type 2 diabetes, the role of AMP-activated protein kinase (AMPK) in the control of insulin secretion is still debatable. Previous studies have used pharmacological activators of limited selectivity and specificity, and none has examined in primary pancreatic beta cells the actions of the latest generation of highly potent and specific activators that act via the allosteric drug and metabolite (ADaM) site. METHODS: AMPK was activated acutely in islets isolated from C57BL6/J mice, and in an EndoC-ßH3 cell line, using three structurally distinct ADaM site activators (991, PF-06409577 and RA089), with varying selectivity for ß1- vs ß2-containing complexes. Mouse lines expressing a gain-of-function mutation in the γ1 AMPK subunit (D316a) were generated to examine the effects of chronic AMPK stimulation in the whole body, or selectively in the beta cell. RESULTS: Acute (1.5 h) treatment of wild-type mouse islets with 991, PF-06409577 or RA089 robustly stimulated insulin secretion at high glucose concentrations (p<0.01, p<0.05 and p<0.001, respectively), despite a lowering of glucose-induced intracellular free Ca2+ dynamics in response to 991 (AUC, p<0.05) and to RA089 at the highest dose (25 µmol/l) at 5.59 min (p<0.05). Although abolished in the absence of AMPK, the effects of 991 were observed in the absence of the upstream kinase, liver kinase B1, further implicating 'amplifying' pathways. In marked contrast, chronic activation of AMPK, either globally or selectively in the beta cell, achieved using a gain-of-function mutant, impaired insulin release in vivo (p<0.05 at 15 min following i.p. injection of 3 mmol/l glucose) and in vitro (p<0.01 following incubation of islets with 17 mmol/l glucose), and lowered glucose tolerance (p<0.001). CONCLUSIONS/INTERPRETATION: AMPK activation exerts complex, time-dependent effects on insulin secretion. These observations should inform the design and future clinical use of AMPK modulators.


Asunto(s)
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Ratones
2.
Diabetes Obes Metab ; 24(11): 2090-2101, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-35676825

RESUMEN

AIMS: To describe the in vitro characteristics and antidiabetic in vivo efficacy of the novel glucagon-like peptide-1 receptor agonist (GLP-1RA) GL0034. MATERIALS AND METHODS: Glucagon-like peptide-1 receptor (GLP-1R) kinetic binding parameters, cyclic adenosine monophosphate (cAMP) signalling, endocytosis and recycling were measured using HEK293 and INS-1832/3 cells expressing human GLP-1R. Insulin secretion was measured in vitro using INS-1832/3 cells, mouse islets and human islets. Chronic administration studies to evaluate weight loss and glycaemic effects were performed in db/db and diet-induced obese mice. RESULTS: Compared to the leading GLP-1RA semaglutide, GL0034 showed increased binding affinity and potency-driven bias in favour of cAMP over GLP-1R endocytosis and ß-arrestin-2 recruitment. Insulin secretory responses were similar for both ligands. GL0034 (6 nmol/kg) led to at least as much weight loss and lowering of blood glucose as did semaglutide at a higher dose (14 nmol/kg). CONCLUSIONS: GL0034 is a G protein-biased agonist that shows powerful antidiabetic effects in mice, and may serve as a promising new GLP-1RA for obese patients with type 2 diabetes.


Asunto(s)
Diabetes Mellitus Tipo 2 , Insulinas , Adenosina Monofosfato , Animales , Glucemia , AMP Cíclico/metabolismo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Receptor del Péptido 1 Similar al Glucagón/agonistas , Células HEK293 , Humanos , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Ligandos , Ratones , Pérdida de Peso , beta-Arrestinas/metabolismo
3.
Diabetologia ; 64(1): 129-141, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33068125

RESUMEN

AIMS/HYPOTHESIS: Transcription factor 7-like 2 (TCF7L2) is a downstream effector of the Wnt/ß-catenin signalling pathway implicated in type 2 diabetes risk through genome-wide association studies. Although its expression is critical for adipocyte development, the potential roles of changes in adipose tissue TCF7L2 levels in diabetes risk are poorly defined. Here, we investigated whether forced changes in Tcf7l2 expression in adipocytes affect whole body glucose or lipid metabolism and crosstalk between disease-relevant tissues. METHODS: Tcf7l2 was selectively ablated in mature adipocytes in C57BL/6J mice using Cre recombinase under Adipoq promoter control to recombine Tcf7l2 alleles floxed at exon 1 (referred to as aTCF7L2 mice). aTCF7L2 mice were fed normal chow or a high-fat diet for 12 weeks. Glucose and insulin sensitivity, as well as beta cell function, were assessed in vivo and in vitro. Levels of circulating NEFA, selected hormones and adipokines were measured using standard assays. RESULTS: Reduced TCF7L2 expression in adipocytes altered glucose tolerance and insulin secretion in male but not in female mice. Thus, on a normal chow diet, male heterozygote knockout mice (aTCF7L2het) exhibited impaired glucose tolerance at 16 weeks (p = 0.03) and increased fat mass (1.4 ± 0.1-fold, p = 0.007) but no changes in insulin secretion. In contrast, male homozygote knockout (aTCF7L2hom) mice displayed normal body weight but impaired oral glucose tolerance at 16 weeks (p = 0.0001). These changes were mechanistically associated with impaired in vitro glucose-stimulated insulin secretion (decreased 0.5 ± 0.1-fold vs control mice, p = 0.02) and decreased levels of the incretins glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide (0.6 ± 0.1-fold and 0.4 ± 0.1-fold vs control mice, p = 0.04 and p < 0.0001, respectively). Circulating levels of plasma NEFA and fatty acid binding protein 4 were increased by 1.3 ± 0.1-fold and 1.8 ± 0.3-fold vs control mice (p = 0.03 and p = 0.05, respectively). Following exposure to a high-fat diet for 12 weeks, male aTCF7L2hom mice exhibited reduced in vivo glucose-stimulated insulin secretion (0.5 ± 0.1-fold vs control mice, p = 0.02). CONCLUSIONS/INTERPRETATION: Loss of Tcf7l2 gene expression selectively in adipocytes leads to a sexually dimorphic phenotype, with impairments not only in adipocytes, but also in pancreatic islet and enteroendocrine cells in male mice only. Our findings suggest novel roles for adipokines and incretins in the effects of diabetes-associated variants in TCF7L2, and further illuminate the roles of TCF7L2 in glucose homeostasis and diabetes risk. Graphical abstract.


Asunto(s)
Adipocitos/metabolismo , Intolerancia a la Glucosa/genética , Metabolismo de los Lípidos/genética , Proteína 2 Similar al Factor de Transcripción 7/genética , Proteína 2 Similar al Factor de Transcripción 7/fisiología , Animales , Composición Corporal/genética , Proteínas de Unión a Ácidos Grasos/sangre , Ácidos Grasos no Esterificados/sangre , Femenino , Expresión Génica , Glucosa/farmacología , Incretinas/sangre , Secreción de Insulina/efectos de los fármacos , Secreción de Insulina/fisiología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Integrasas/genética , Integrasas/fisiología , Metabolismo de los Lípidos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados
4.
Am J Hum Genet ; 100(2): 238-256, 2017 02 02.
Artículo en Inglés | MEDLINE | ID: mdl-28132686

RESUMEN

Genetic variants near ARAP1 (CENTD2) and STARD10 influence type 2 diabetes (T2D) risk. The risk alleles impair glucose-induced insulin secretion and, paradoxically but characteristically, are associated with decreased proinsulin:insulin ratios, indicating improved proinsulin conversion. Neither the identity of the causal variants nor the gene(s) through which risk is conferred have been firmly established. Whereas ARAP1 encodes a GTPase activating protein, STARD10 is a member of the steroidogenic acute regulatory protein (StAR)-related lipid transfer protein family. By integrating genetic fine-mapping and epigenomic annotation data and performing promoter-reporter and chromatin conformational capture (3C) studies in ß cell lines, we localize the causal variant(s) at this locus to a 5 kb region that overlaps a stretch-enhancer active in islets. This region contains several highly correlated T2D-risk variants, including the rs140130268 indel. Expression QTL analysis of islet transcriptomes from three independent subject groups demonstrated that T2D-risk allele carriers displayed reduced levels of STARD10 mRNA, with no concomitant change in ARAP1 mRNA levels. Correspondingly, ß-cell-selective deletion of StarD10 in mice led to impaired glucose-stimulated Ca2+ dynamics and insulin secretion and recapitulated the pattern of improved proinsulin processing observed at the human GWAS signal. Conversely, overexpression of StarD10 in the adult ß cell improved glucose tolerance in high fat-fed animals. In contrast, manipulation of Arap1 in ß cells had no impact on insulin secretion or proinsulin conversion in mice. This convergence of human and murine data provides compelling evidence that the T2D risk associated with variation at this locus is mediated through reduction in STARD10 expression in the ß cell.


Asunto(s)
Diabetes Mellitus Tipo 2/genética , Insulina/metabolismo , Fosfoproteínas/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Alelos , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Clonación Molecular , Diabetes Mellitus Tipo 2/sangre , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Regulación de la Expresión Génica , Variación Genética , Homeostasis , Humanos , Insulina/sangre , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Hígado/metabolismo , Ratones , Proinsulina/sangre , Proinsulina/metabolismo , Sitios de Carácter Cuantitativo , Transcriptoma
5.
Br J Nutr ; 124(3): 247-255, 2020 08 14.
Artículo en Inglés | MEDLINE | ID: mdl-32122411

RESUMEN

Preliminary evidence has suggested that high-fat diets (HFD) enriched with SFA, but not MUFA, promote hyperinsulinaemia and pancreatic hypertrophy with insulin resistance. The objective of this study was to determine whether the substitution of dietary MUFA within a HFD could attenuate the progression of pancreatic islet dysfunction seen with prolonged SFA-HFD. For 32 weeks, C57BL/6J mice were fed either: (1) low-fat diet, (2) SFA-HFD or (3) SFA-HFD for 16 weeks, then switched to MUFA-HFD for 16 weeks (SFA-to-MUFA-HFD). Fasting insulin was assessed throughout the study; islets were isolated following the intervention. Substituting SFA with MUFA-HFD prevented the progression of hyperinsulinaemia observed in SFA-HFD mice (P < 0·001). Glucose-stimulated insulin secretion from isolated islets was reduced by SFA-HFD, yet not fully affected by SFA-to-MUFA-HFD. Markers of ß-cell identity (Ins2, Nkx6.1, Ngn3, Rfx6, Pdx1 and Pax6) were reduced, and islet inflammation was increased (IL-1ß, 3·0-fold, P = 0·007; CD68, 2·9-fold, P = 0·001; Il-6, 1·1-fold, P = 0·437) in SFA-HFD - effects not seen with SFA-to-MUFA-HFD. Switching to MUFA-HFD can partly attenuate the progression of SFA-HFD-induced hyperinsulinaemia, pancreatic inflammation and impairments in ß-cell function. While further work is required from a mechanistic perspective, dietary fat may mediate its effect in an IL-1ß-AMP-activated protein kinase α1-dependent fashion. Future work should assess the potential translation of the modulation of metabolic inflammation in man.


Asunto(s)
Dieta Alta en Grasa/métodos , Grasas de la Dieta/farmacología , Ácidos Grasos Monoinsaturados/farmacología , Ácidos Grasos/farmacología , Hiperinsulinismo/dietoterapia , Animales , Modelos Animales de Enfermedad , Resistencia a la Insulina/fisiología , Islotes Pancreáticos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Páncreas/efectos de los fármacos
6.
J Biol Chem ; 293(36): 14178-14189, 2018 09 07.
Artículo en Inglés | MEDLINE | ID: mdl-29967064

RESUMEN

Variants in the transcription factor-7-like 2 (TCF7L2/TCF4) gene, involved in Wnt signaling, are associated with type 2 diabetes. Loss of Tcf7l2 selectively from the ß cell in mice has previously been shown to cause glucose intolerance and to lower ß cell mass. Deletion of the tumor suppressor liver kinase B1 (LKB1/STK11) leads to ß cell hyperplasia and enhanced glucose-stimulated insulin secretion, providing a convenient genetic model for increased ß cell growth and function. The aim of this study was to explore the possibility that Tcf7l2 may be required for the effects of Lkb1 deletion on insulin secretion in the mouse ß cell. Mice bearing floxed Lkb1 and/or Tcf7l2 alleles were bred with knockin mice bearing Cre recombinase inserted at the Ins1 locus (Ins1Cre), allowing highly ß cell-selective deletion of either or both genes. Oral glucose tolerance was unchanged by the further deletion of a single Tcf7l2 allele in these cells. By contrast, mice lacking both Tcf7l2 alleles on this background showed improved oral glucose tolerance and insulin secretion in vivo and in vitro compared with mice lacking a single Tcf7l2 allele. Biallelic Tcf7l2 deletion also enhanced ß cell proliferation, increased ß cell mass, and caused changes in polarity as revealed by the "rosette-like" arrangement of ß cells. Tcf7l2 deletion also increased signaling by mammalian target of rapamycin (mTOR), augmenting phospho-ribosomal S6 levels. We identified a novel signaling mechanism through which a modifier gene, Tcf7l2, lies on a pathway through which LKB1 acts in the ß cell to restrict insulin secretion.


Asunto(s)
Secreción de Insulina , Células Secretoras de Insulina/citología , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Proteína 2 Similar al Factor de Transcripción 7/fisiología , Proteínas Quinasas Activadas por AMP , Animales , Proliferación Celular , Diabetes Mellitus Tipo 2 , Ratones , Proteínas Serina-Treonina Quinasas
7.
Int J Obes (Lond) ; 43(3): 556-566, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30006585

RESUMEN

BACKGROUND/OBJECTIVES: The increased prevalence of obesity has prompted great strides in our understanding of specific adipose depots and their involvement in cardio-metabolic health. However, the impact of obesity on dermal white adipose tissue (dWAT) and dermal microvascular functionality remains unclear. This study aimed to investigate the temporal changes that occur in dWAT and dermal microvascular functionality during the development of diet-induced obesity and type 2 diabetes in mice. METHODS: Metabolic phenotyping of a murine model of hypercaloric diet (HCD)-induced obesity and type 2 diabetes was performed at three time points that reflected three distinct stages of disease development; 2 weeks of HCD-overweight-metabolically healthy, 4 weeks of HCD-obese-prediabetic and 12 weeks of HCD-obese-type 2 diabetic mice. Expansion of dWAT was characterized histologically, and changes in dermal microvascular reactivity were assessed in response to pressure and the vasodilators SNP and Ach. RESULTS: HCD resulted in a progressive expansion of dWAT and increased expression of pro-inflammatory markers (IL1ß and COX-2). Impairments in pressure-induced (PIV) and Ach-induced (endothelium-dependent) vasodilation occurred early, in overweight-metabolically healthy mice. Residual vasodilatory responses were NOS-independent but sensitive to COX inhibition. These changes were associated with reductions in NO and adiponectin bioavailability, and rescued by exogenous adiponectin or hyperinsulinemia. Obese-prediabetic mice continued to exhibit impaired Ach-dependent vasodilation but PIV appeared normalized. This normalization coincided with elevated endogenous adiponectin and insulin levels, and was sensitive to NOS, COX and PI3K, inhibition. In obese-type 2 diabetic mice, both Ach-stimulated and pressure-induced vasodilatory responses were increased through enhanced COX-2-dependent prostaglandin response. CONCLUSIONS: We demonstrate that the development of obesity, metabolic dysfunction and type 2 diabetes, in HCD-fed mice, is accompanied by increased dermal adiposity and associated metaflammation in dWAT. Importantly, these temporal changes are also linked to disease stage-specific dermal microvascular reactivity, which may reflect adaptive mechanisms driven by metaflammation.


Asunto(s)
Tejido Adiposo Blanco , Diabetes Mellitus Tipo 2/fisiopatología , Inflamación , Obesidad/fisiopatología , Piel , Adiponectina/metabolismo , Tejido Adiposo Blanco/irrigación sanguínea , Tejido Adiposo Blanco/metabolismo , Tejido Adiposo Blanco/patología , Tejido Adiposo Blanco/fisiopatología , Animales , Citocinas/metabolismo , Diabetes Mellitus Experimental , Inflamación/metabolismo , Inflamación/patología , Inflamación/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Microvasos/metabolismo , Microvasos/patología , Microvasos/fisiopatología , Piel/irrigación sanguínea , Piel/metabolismo , Piel/fisiopatología , Vasodilatación/fisiología
8.
FASEB J ; 32(5): 2587-2600, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29269398

RESUMEN

AMPK is a critical energy sensor and target for widely used antidiabetic drugs. In ß cells, elevated glucose concentrations lower AMPK activity, and the ablation of both catalytic subunits [ß-cell-specific AMPK double-knockout (ßAMPKdKO) mice] impairs insulin secretion in vivo and ß-cell identity. MicroRNAs (miRNAs) are small RNAs that silence gene expression that are essential for pancreatic ß-cell function and identity and altered in diabetes. Here, we have explored the miRNAs acting downstream of AMPK in mouse and human ß cells. We identified 14 down-regulated and 9 up-regulated miRNAs in ßAMPKdKO vs. control islets. Gene ontology analysis of targeted transcripts revealed enrichment in pathways important for ß-cell function and identity. The most down-regulated miRNA was miR-184 (miR-184-3p), an important regulator of ß-cell function and compensatory expansion that is controlled by glucose and reduced in diabetes. We demonstrate that AMPK is a potent regulator and an important mediator of the negative effects of glucose on miR-184 expression. Additionally, we reveal sexual dimorphism in miR-184 expression in mouse and human islets. Collectively, these data demonstrate that glucose-mediated changes in AMPK activity are central for the regulation of miR-184 and other miRNAs in islets and provide a link between energy status and gene expression in ß cells.-Martinez-Sanchez, A., Nguyen-Tu, M.-S., Cebola, I., Yavari, A., Marchetti, P., Piemonti, L., de Koning, E., Shapiro, A. M. J., Johnson, P., Sakamoto, K., Smith, D. M., Leclerc, I., Ashrafian, H., Ferrer, J., Rutter, G. A. MiR-184 expression is regulated by AMPK in pancreatic islets.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Diabetes Mellitus/metabolismo , Regulación de la Expresión Génica , Células Secretoras de Insulina/metabolismo , MicroARNs/biosíntesis , Proteínas Quinasas Activadas por AMP/genética , Animales , Línea Celular , Diabetes Mellitus/genética , Diabetes Mellitus/patología , Metabolismo Energético/genética , Femenino , Glucosa/genética , Glucosa/metabolismo , Humanos , Células Secretoras de Insulina/patología , Masculino , Ratones , Ratones Noqueados , MicroARNs/genética , Caracteres Sexuales
9.
J Biol Chem ; 292(21): 8892-8906, 2017 05 26.
Artículo en Inglés | MEDLINE | ID: mdl-28377501

RESUMEN

Heterozygous mutations in the human paired box gene PAX6 lead to impaired glucose tolerance. Although embryonic deletion of the Pax6 gene in mice leads to loss of most pancreatic islet cell types, the functional consequences of Pax6 loss in adults are poorly defined. Here we developed a mouse line in which Pax6 was selectively inactivated in ß cells by crossing animals with floxed Pax6 alleles to mice expressing the inducible Pdx1CreERT transgene. Pax6 deficiency, achieved by tamoxifen injection, caused progressive hyperglycemia. Although ß cell mass was preserved 8 days post-injection, total insulin content and insulin:chromogranin A immunoreactivity were reduced by ∼60%, and glucose-stimulated insulin secretion was eliminated. RNA sequencing and quantitative real-time PCR analyses revealed that, although the expression of key ß cell genes, including Ins2, Slc30a8, MafA, Slc2a2, G6pc2, and Glp1r, was reduced after Pax6 deletion, that of several genes that are usually selectively repressed ("disallowed") in ß cells, including Slc16a1, was increased. Assessed in intact islets, glucose-induced ATP:ADP increases were significantly reduced (p < 0.05) in ßPax6KO versus control ß cells, and the former displayed attenuated increases in cytosolic Ca2+ Unexpectedly, glucose-induced increases in intercellular connectivity were enhanced after Pax6 deletion, consistent with increases in the expression of the glucose sensor glucokinase, but decreases in that of two transcription factors usually expressed in fully differentiated ß-cells, Pdx1 and Nkx6.1, were observed in islet "hub" cells. These results indicate that Pax6 is required for the functional identity of adult ß cells. Furthermore, deficiencies in ß cell glucose sensing are likely to contribute to defective insulin secretion in human carriers of PAX6 mutations.


Asunto(s)
Adenosina Trifosfato/metabolismo , Señalización del Calcio , Calcio/metabolismo , Regulación de la Expresión Génica , Glucosa/metabolismo , Células Secretoras de Insulina/metabolismo , Factor de Transcripción PAX6/biosíntesis , Adenosina Trifosfato/genética , Animales , Humanos , Ratones , Ratones Noqueados , Factor de Transcripción PAX6/genética
10.
Diabetes Obes Metab ; 20(9): 2246-2254, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29748994

RESUMEN

AIMS: To investigate the role of arcuate glucokinase (GK) in the regulation of glucose homeostasis. MATERIALS AND METHODS: A recombinant adeno-associated virus expressing either GK or an antisense GK construct was used to alter GK activity specifically in the hypothalamic arcuate nucleus (arc). GK activity in this nucleus was also increased by stereotactic injection of the GK activator, compound A. The effect of altered arc GK activity on glucose homeostasis was subsequently investigated using glucose and insulin tolerance tests. RESULTS: Increased GK activity specifically within the arc increased insulin secretion and improved glucose tolerance in rats during oral glucose tolerance tests. Decreased GK activity in this nucleus reduced insulin secretion and increased glucose levels during the same tests. Insulin sensitivity was not affected in either case. The effect of arc GK was maintained in a model of type 2 diabetes. CONCLUSIONS: These results demonstrate a role for arc GK in systemic glucose homeostasis.


Asunto(s)
Núcleo Arqueado del Hipotálamo/enzimología , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Tipo 2/enzimología , Glucoquinasa/metabolismo , Glucosa/metabolismo , Secreción de Insulina/fisiología , Animales , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Tipo 2/sangre , Prueba de Tolerancia a la Glucosa , Homeostasis/fisiología , Masculino , Ratas , Ratas Wistar , Ratas Zucker
11.
FASEB J ; 28(11): 4972-85, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-25070369

RESUMEN

Fully differentiated pancreatic ß cells are essential for normal glucose homeostasis in mammals. Dedifferentiation of these cells has been suggested to occur in type 2 diabetes, impairing insulin production. Since chronic fuel excess ("glucotoxicity") is implicated in this process, we sought here to identify the potential roles in ß-cell identity of the tumor suppressor liver kinase B1 (LKB1/STK11) and the downstream fuel-sensitive kinase, AMP-activated protein kinase (AMPK). Highly ß-cell-restricted deletion of each kinase in mice, using an Ins1-controlled Cre, was therefore followed by physiological, morphometric, and massive parallel sequencing analysis. Loss of LKB1 strikingly (2.0-12-fold, E<0.01) increased the expression of subsets of hepatic (Alb, Iyd, Elovl2) and neuronal (Nptx2, Dlgap2, Cartpt, Pdyn) genes, enhancing glutamate signaling. These changes were partially recapitulated by the loss of AMPK, which also up-regulated ß-cell "disallowed" genes (Slc16a1, Ldha, Mgst1, Pdgfra) 1.8- to 3.4-fold (E < 0.01). Correspondingly, targeted promoters were enriched for neuronal (Zfp206; P = 1.3 × 10(-33)) and hypoxia-regulated (HIF1; P = 2.5 × 10(-16)) transcription factors. In summary, LKB1 and AMPK, through only partly overlapping mechanisms, maintain ß-cell identity by suppressing alternate pathways leading to neuronal, hepatic, and other characteristics. Selective targeting of these enzymes may provide a new approach to maintaining ß-cell function in some forms of diabetes.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Células Secretoras de Insulina/enzimología , Insulina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Diabetes Mellitus Tipo 2/metabolismo , Ratones Endogámicos C57BL , Transducción de Señal/fisiología
13.
Microvasc Res ; 90: 138-43, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24120748

RESUMEN

PURPOSE: The role of obesity in the appearance of skin pressure ulcers remains controversial. The aim of the present study was to evaluate blood perfusion and related lesions after skin compression in obese mice. METHODS: Sixty C57BL6 male mice were randomly assigned to a control or hypercalorific diet (HCD) for 2, 4 and 12weeks. Skin compression was induced by a magnetic force of 11 kPa overlying a subcutaneous metal plate and applied for 4h. Skin perfusion was examined using laser Doppler imaging before skin compression, immediately after compression release and 24h later. 24h after magnet removal, skin injuries were determined by photography. RESULTS: A heterogeneous distribution of blood perfusion was observed using the colour-coded map of the skin perfusion on the compressed area. At 24-h post-compression release, 60% to 75% of the compressed area was ischaemic in the 2-week HCD group and in all the control groups compared to 35% in the 4- and 15% in the 12-week HCD groups. The lowest occurrence of skin lesion seen as skin redness or pressure-sores was observed in the 12- week HCD group (4%) compared to about 12% in either the control or the 2- and 4-week HCD groups. CONCLUSIONS: This study suggests that there was no clear relationship between the extent of ischaemia and skin lesion occurrence after skin compression in short-duration obese mice. In contrast, it appears that long-duration obesity could reduce both ischaemia and skin lesions in response to skin compression through changes in skin structure.


Asunto(s)
Isquemia/fisiopatología , Microcirculación , Microvasos/fisiopatología , Obesidad/fisiopatología , Úlcera por Presión/fisiopatología , Piel/irrigación sanguínea , Animales , Velocidad del Flujo Sanguíneo , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Ingestión de Energía , Isquemia/etiología , Isquemia/metabolismo , Isquemia/prevención & control , Flujometría por Láser-Doppler , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/etiología , Obesidad/metabolismo , Presión , Úlcera por Presión/etiología , Úlcera por Presión/metabolismo , Úlcera por Presión/prevención & control , Flujo Sanguíneo Regional , Factores de Tiempo
14.
Diabetes ; 71(7): 1525-1545, 2022 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-35476777

RESUMEN

Impaired pancreatic ß-cell function and insulin secretion are hallmarks of type 2 diabetes. miRNAs are short, noncoding RNAs that silence gene expression vital for the development and function of ß cells. We have previously shown that ß cell-specific deletion of the important energy sensor AMP-activated protein kinase (AMPK) results in increased miR-125b-5p levels. Nevertheless, the function of this miRNA in ß cells is unclear. We hypothesized that miR-125b-5p expression is regulated by glucose and that this miRNA mediates some of the deleterious effects of hyperglycemia in ß cells. Here, we show that islet miR-125b-5p expression is upregulated by glucose in an AMPK-dependent manner and that short-term miR-125b-5p overexpression impairs glucose-stimulated insulin secretion (GSIS) in the mouse insulinoma MIN6 cells and in human islets. An unbiased, high-throughput screen in MIN6 cells identified multiple miR-125b-5p targets, including the transporter of lysosomal hydrolases M6pr and the mitochondrial fission regulator Mtfp1. Inactivation of miR-125b-5p in the human ß-cell line EndoCß-H1 shortened mitochondria and enhanced GSIS, whereas mice overexpressing miR-125b-5p selectively in ß cells (MIR125B-Tg) were hyperglycemic and glucose intolerant. MIR125B-Tg ß cells contained enlarged lysosomal structures and had reduced insulin content and secretion. Collectively, we identify miR-125b as a glucose-controlled regulator of organelle dynamics that modulates insulin secretion.


Asunto(s)
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , MicroARNs , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Glucosa/farmacología , Humanos , Células Secretoras de Insulina/metabolismo , Ratones , MicroARNs/genética , MicroARNs/metabolismo
15.
JCI Insight ; 5(8)2020 03 31.
Artículo en Inglés | MEDLINE | ID: mdl-32229720

RESUMEN

Glucokinase (GK) is highly expressed in the hypothalamic paraventricular nucleus (PVN); however, its role is currently unknown. We found that GK in the PVN acts as part of a glucose-sensing mechanism within the PVN that regulates glucose homeostasis by controlling glucagon-like peptide 1 (GLP-1) release. GLP-1 is released from enteroendocrine L cells in response to oral glucose. Here we identify a brain mechanism critical to the release of GLP-1 in response to oral glucose. We show that increasing expression of GK or injection of glucose into the PVN increases GLP-1 release in response to oral glucose. On the contrary, decreasing expression of GK or injection of nonmetabolizable glucose into the PVN prevents GLP-1 release. Our results demonstrate that gluco-sensitive GK neurons in the PVN are critical to the response to oral glucose and subsequent release of GLP-1.


Asunto(s)
Péptido 1 Similar al Glucagón/genética , Glucosa/farmacología , Núcleo Hipotalámico Paraventricular/metabolismo , Animales , Glucoquinasa/metabolismo , Masculino , Ratas , Ratas Endogámicas WF
16.
Methods Mol Biol ; 1732: 413-431, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29480490

RESUMEN

The role of the energy sensor AMPK-activated protein kinase (AMPK) in the insulin-secreting ß-cell remains unclear and a subject of intense research. With this chapter, we aim to provide a detailed description of the methods that our group routinely applies to the study of AMPK function in mouse and human pancreatic islets. Thus, we provide detailed protocols to isolate and/or culture mouse and human islets, to modulate and measure AMPK activity in isolated islets, and to evaluate its impact on islet function.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Activación Enzimática/efectos de los fármacos , Pruebas de Enzimas/métodos , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Línea Celular , Activadores de Enzimas/farmacología , Pruebas de Enzimas/instrumentación , Glucosa/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Cultivo Primario de Células , Ribonucleótidos/farmacología
17.
Sci Rep ; 7(1): 291, 2017 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-28331198

RESUMEN

Photopharmacology describes the use of light to precisely deliver drug activity in space and time. Such approaches promise to improve drug specificity by reducing off-target effects. As a proof-of-concept, we have subjected the fourth generation photoswitchable sulfonylurea JB253 to comprehensive toxicology assessment, including mutagenicity and maximum/repeated tolerated dose studies, as well as in vivo testing in rodents. Here, we show that JB253 is well-tolerated with minimal mutagenicity and can be used to optically-control glucose homeostasis in anesthetized mice following delivery of blue light to the pancreas. These studies provide the first demonstration that photopharmacology may one day be applicable to the light-guided treatment of type 2 diabetes and other metabolic disease states in vivo in humans.


Asunto(s)
Diabetes Mellitus/tratamiento farmacológico , Hipoglucemiantes/administración & dosificación , Fármacos Fotosensibilizantes/administración & dosificación , Compuestos de Sulfonilurea/administración & dosificación , Animales , Glucemia , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Homeostasis , Hipoglucemiantes/efectos adversos , Ratones , Fármacos Fotosensibilizantes/efectos adversos , Compuestos de Sulfonilurea/efectos adversos , Resultado del Tratamiento
18.
Diabetes ; 65(5): 1268-82, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-26861785

RESUMEN

Encoding acyl-CoA thioesterase-7 (Acot7) is one of ∼60 genes expressed ubiquitously across tissues but relatively silenced, or disallowed, in pancreatic ß-cells. The capacity of ACOT7 to hydrolyze long-chain acyl-CoA esters suggests potential roles in ß-oxidation, lipid biosynthesis, signal transduction, or insulin exocytosis. We explored the physiological relevance of ß-cell-specific Acot7 silencing by re-expressing ACOT7 in these cells. ACOT7 overexpression in clonal MIN6 and INS1(832/13) ß-cells impaired insulin secretion in response to glucose plus fatty acids. Furthermore, in a panel of transgenic mouse lines, we demonstrate that overexpression of mitochondrial ACOT7 selectively in the adult ß-cell reduces glucose tolerance dose dependently and impairs glucose-stimulated insulin secretion. By contrast, depolarization-induced secretion was unaffected, arguing against a direct action on the exocytotic machinery. Acyl-CoA levels, ATP/ADP increases, membrane depolarization, and Ca(2+) fluxes were all markedly reduced in transgenic mouse islets, whereas glucose-induced oxygen consumption was unchanged. Although glucose-induced increases in ATP/ADP ratio were similarly lowered after ACOT7 overexpression in INS1(832/13) cells, changes in mitochondrial membrane potential were unaffected, consistent with an action of Acot7 to increase cellular ATP consumption. Because Acot7 mRNA levels are increased in human islets in type 2 diabetes, inhibition of the enzyme might provide a novel therapeutic strategy.


Asunto(s)
Regulación hacia Abajo , Ácidos Grasos no Esterificados/metabolismo , Regulación Enzimológica de la Expresión Génica , Glucosa/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Palmitoil-CoA Hidrolasa/metabolismo , Animales , Señalización del Calcio , Línea Celular Tumoral , Células Clonales , Femenino , Intolerancia a la Glucosa/enzimología , Intolerancia a la Glucosa/metabolismo , Intolerancia a la Glucosa/patología , Secreción de Insulina , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/patología , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Especificidad de Órganos , Palmitoil-CoA Hidrolasa/genética , Ratas , Proteínas Recombinantes/metabolismo , Caracteres Sexuales , Técnicas de Cultivo de Tejidos , Regulación hacia Arriba
19.
Cell Metab ; 23(5): 821-36, 2016 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-27133129

RESUMEN

Despite significant advances in our understanding of the biology determining systemic energy homeostasis, the treatment of obesity remains a medical challenge. Activation of AMP-activated protein kinase (AMPK) has been proposed as an attractive strategy for the treatment of obesity and its complications. AMPK is a conserved, ubiquitously expressed, heterotrimeric serine/threonine kinase whose short-term activation has multiple beneficial metabolic effects. Whether these translate into long-term benefits for obesity and its complications is unknown. Here, we observe that mice with chronic AMPK activation, resulting from mutation of the AMPK γ2 subunit, exhibit ghrelin signaling-dependent hyperphagia, obesity, and impaired pancreatic islet insulin secretion. Humans bearing the homologous mutation manifest a congruent phenotype. Our studies highlight that long-term AMPK activation throughout all tissues can have adverse metabolic consequences, with implications for pharmacological strategies seeking to chronically activate AMPK systemically to treat metabolic disease.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Células Secretoras de Insulina/enzimología , Células Secretoras de Insulina/patología , Obesidad/enzimología , Adiposidad/genética , Adulto , Envejecimiento/patología , Proteína Relacionada con Agouti/metabolismo , Animales , Núcleo Arqueado del Hipotálamo/metabolismo , Metabolismo Energético/genética , Activación Enzimática , Conducta Alimentaria , Femenino , Heterocigoto , Humanos , Hiperfagia/complicaciones , Hiperfagia/enzimología , Hiperfagia/genética , Hiperfagia/patología , Hipotálamo/metabolismo , Insulina/metabolismo , Masculino , Ratones , Mitocondrias/metabolismo , Mutación/genética , Neuronas/metabolismo , Obesidad/sangre , Obesidad/complicaciones , Obesidad/patología , Fosforilación Oxidativa , Receptores de Ghrelina/metabolismo , Ribosomas/metabolismo , Transducción de Señal/genética , Transcriptoma/genética , Regulación hacia Arriba/genética
20.
Mol Endocrinol ; 29(7): 1067-79, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-26038943

RESUMEN

Pancreatic ß-cells are the body's sole source of circulating insulin and essential for the maintenance of blood glucose homeostasis. Levels of up to 66 "disallowed" genes, which are strongly expressed and play housekeeping roles in most other mammalian tissues, are unusually low in ß-cells. The molecular mechanisms involved in repressing these genes are largely unknown. Here, we explore the role in gene disallowance of microRNAs (miRNAs), a type of small noncoding RNAs that silence gene expression at the posttranscriptional level and are essential for ß-cell development and function. To selectively deplete miRNAs from adult ß-cells, the miRNA-processing enzyme DICER was inactivated by deletion of the RNase III domain with a tamoxifen-inducible Pdx1CreER transgene. In this model, ß-cell dysfunction was apparent 2 weeks after recombination and preceded a decrease in insulin content and loss of ß-cell mass. Of the 14 disallowed genes studied, quantitative RT-quantitative real-time PCR revealed that 6 genes (Fcgrt, Igfbp4, Maf, Oat, Pdgfra, and Slc16a1) were up-regulated (1.4- to 2.1-fold, P < .05) at this early stage. Expression of luciferase constructs bearing the 3'-untranslated regions of the corresponding mRNAs in wild-type or DICER-null ß-cells demonstrated that Fcgrt, Oat, and Pdgfra are miRNA direct targets. We thus reveal a role for miRNAs in the regulation of disallowed genes in ß-cells and provide evidence for a novel means through which noncoding RNAs control the functional identity of these cells independently of actions on ß-cell mass.


Asunto(s)
Células Secretoras de Insulina/metabolismo , MicroARNs/metabolismo , Ribonucleasa III/metabolismo , Animales , Apoptosis/efectos de los fármacos , Eliminación de Gen , Glucosa/farmacología , Intolerancia a la Glucosa/complicaciones , Intolerancia a la Glucosa/genética , Proteínas de Homeodominio/metabolismo , Hiperglucemia/complicaciones , Hiperglucemia/genética , Insulina/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Integrasas/metabolismo , Ratones Endogámicos C57BL , MicroARNs/genética , Ribonucleasa III/deficiencia , Tamoxifeno/administración & dosificación , Tamoxifeno/farmacología , Transactivadores/metabolismo , Regulación hacia Arriba/efectos de los fármacos
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