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1.
Diabetologia ; 63(2): 313-323, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31732790

RESUMO

AIMS/HYPOTHESIS: Progressive decline in functional beta cell mass is central to the development of type 2 diabetes. Elevated serum levels of extracellular nicotinamide phosphoribosyltransferase (eNAMPT) are associated with beta cell failure in type 2 diabetes and eNAMPT immuno-neutralisation improves glucose tolerance in mouse models of diabetes. Despite this, the effects of eNAMPT on functional beta cell mass are poorly elucidated, with some studies having separately reported beta cell-protective effects of eNAMPT. eNAMPT exists in structurally and functionally distinct monomeric and dimeric forms. Dimerisation is essential for the NAD-biosynthetic capacity of NAMPT. Monomeric eNAMPT does not possess NAD-biosynthetic capacity and may exert distinct NAD-independent effects. This study aimed to fully characterise the structure-functional effects of eNAMPT on pancreatic beta cell functional mass and to relate these to beta cell failure in type 2 diabetes. METHODS: CD-1 mice and serum from obese humans who were without diabetes, with impaired fasting glucose (IFG) or with type 2 diabetes (from the Body Fat, Surgery and Hormone [BodyFatS&H] study) or with or at risk of developing type 2 diabetes (from the VaSera trial) were used in this study. We generated recombinant wild-type and monomeric eNAMPT to explore the effects of eNAMPT on functional beta cell mass in isolated mouse and human islets. Beta cell function was determined by static and dynamic insulin secretion and intracellular calcium microfluorimetry. NAD-biosynthetic capacity of eNAMPT was assessed by colorimetric and fluorescent assays and by native mass spectrometry. Islet cell number was determined by immunohistochemical staining for insulin, glucagon and somatostatin, with islet apoptosis determined by caspase 3/7 activity. Markers of inflammation and beta cell identity were determined by quantitative reverse transcription PCR. Total, monomeric and dimeric eNAMPT and nicotinamide mononucleotide (NMN) were evaluated by ELISA, western blot and fluorometric assay using serum from non-diabetic, glucose intolerant and type 2 diabetic individuals. RESULTS: eNAMPT exerts bimodal and concentration- and structure-functional-dependent effects on beta cell functional mass. At low physiological concentrations (~1 ng/ml), as seen in serum from humans without diabetes, eNAMPT enhances beta cell function through NAD-dependent mechanisms, consistent with eNAMPT being present as a dimer. However, as eNAMPT concentrations rise to ~5 ng/ml, as in type 2 diabetes, eNAMPT begins to adopt a monomeric form and mediates beta cell dysfunction, reduced beta cell identity and number, increased alpha cell number and increased apoptosis, through NAD-independent proinflammatory mechanisms. CONCLUSIONS/INTERPRETATION: We have characterised a novel mechanism of beta cell dysfunction in type 2 diabetes. At low physiological levels, eNAMPT exists in dimer form and maintains beta cell function and identity through NAD-dependent mechanisms. However, as eNAMPT levels rise, as in type 2 diabetes, structure-functional changes occur resulting in marked elevation of monomeric eNAMPT, which induces a diabetic phenotype in pancreatic islets. Strategies to selectively target monomeric eNAMPT could represent promising therapeutic strategies for the treatment of type 2 diabetes.


Assuntos
Citocinas/sangue , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/fisiopatologia , Nicotinamida Fosforribosiltransferase/sangue , Nicotinamida Fosforribosiltransferase/metabolismo , Animais , Células Cultivadas , Diabetes Mellitus Tipo 2/metabolismo , Glucagon/sangue , Glucagon/metabolismo , Humanos , Immunoblotting , Secreção de Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Masculino , Espectrometria de Massas , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Somatostatina/sangue , Somatostatina/metabolismo , Relação Estrutura-Atividade
2.
Diabetologia ; 59(11): 2477-2486, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27541013

RESUMO

AIMS/HYPOTHESIS: Serum extracellular nicotinamide phosphoribosyltransferase (eNAMPT) concentrations are elevated in type 2 diabetes. However, the relationship between abnormally elevated serum eNAMPT and type 2 diabetes pathophysiology is unclear. eNAMPT circulates in functionally and structurally distinct monomeric and dimeric forms. Dimeric eNAMPT promotes NAD biosynthesis. The role of eNAMPT-monomer is unclear but it may have NAD-independent proinflammatory effects. However, studies of eNAMPT in type 2 diabetes have not distinguished between monomeric and dimeric forms. Since type 2 diabetes is characterised by chronic inflammation, we hypothesised a selective NAD-independent role for eNAMPT-monomer in type 2 diabetes. METHODS: Two mouse models were used to examine the role of eNAMPT-monomer in type 2 diabetes; (1) a mouse model of diabetes fed a high-fat diet (HFD) for 10 weeks received i.p. injections with an anti-monomeric-eNAMPT antibody; and (2) lean non-diabetic mice received i.p. injections with recombinant monomeric eNAMPT daily for 14 days. RESULTS: Serum monomeric eNAMPT levels were elevated in HFD-fed mouse models of diabetes, whilst eNAMPT-dimer levels were unchanged. eNAMPT-monomer neutralisation in HFD-fed mice resulted in lower blood glucose levels, amelioration of impaired glucose tolerance (IGT) and whole-body insulin resistance, improved pancreatic islet function, and reduced inflammation. These effects were maintained for at least 3 weeks post-treatment. eNAMPT-monomer administration induced a diabetic phenotype in mice, characterised by elevated blood glucose, IGT, impaired pancreatic insulin secretion and the presence of systemic and tissue inflammation, without changes in NAD levels. CONCLUSIONS/INTERPRETATION: We demonstrate that elevation of monomeric-eNAMPT plays an important role in the pathogenesis of diet-induced diabetes via proinflammatory mechanisms. These data provide proof-of-concept evidence that the eNAMPT-monomer represents a potential therapeutic target for type 2 diabetes.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Nicotinamida Fosforribosiltransferase/metabolismo , Animais , Anticorpos/uso terapêutico , Linhagem Celular , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Dieta Hiperlipídica/efeitos adversos , Técnicas In Vitro , Insulina/metabolismo , Resistência à Insulina/fisiologia , Ilhotas Pancreáticas/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nicotinamida Fosforribosiltransferase/sangue , Nicotinamida Fosforribosiltransferase/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
3.
J Biol Chem ; 290(34): 20934-20946, 2015 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-26139601

RESUMO

The tumor suppressor liver kinase B1 (LKB1) is an important regulator of pancreatic ß cell biology. LKB1-dependent phosphorylation of distinct AMPK (adenosine monophosphate-activated protein kinase) family members determines proper ß cell polarity and restricts ß cell size, total ß cell mass, and glucose-stimulated insulin secretion (GSIS). However, the full spectrum of LKB1 effects and the mechanisms involved in the secretory phenotype remain incompletely understood. We report here that in the absence of LKB1 in ß cells, GSIS is dramatically and persistently improved. The enhancement is seen both in vivo and in vitro and cannot be explained by altered cell polarity, increased ß cell number, or increased insulin content. Increased secretion does require membrane depolarization and calcium influx but appears to rely mostly on a distal step in the secretion pathway. Surprisingly, enhanced GSIS is seen despite profound defects in mitochondrial structure and function in LKB1-deficient ß cells, expected to greatly diminish insulin secretion via the classic triggering pathway. Thus LKB1 is essential for mitochondrial homeostasis in ß cells and in parallel is a powerful negative regulator of insulin secretion. This study shows that ß cells can be manipulated to enhance GSIS to supra-normal levels even in the face of defective mitochondria and without deterioration over months.


Assuntos
Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Mitocôndrias/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Regulação da Expressão Gênica , Glucose/farmacologia , Ácido Glutâmico/metabolismo , Humanos , Insulina/agonistas , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/patologia , Camundongos , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Fosforilação , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Tamoxifeno/toxicidade , Técnicas de Cultura de Tecidos
4.
FASEB J ; 28(11): 4972-85, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25070369

RESUMO

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.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Células Secretoras de Insulina/enzimologia , Insulina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Diabetes Mellitus Tipo 2/metabolismo , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologia
5.
Free Radic Biol Med ; 175: 65-79, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34455039

RESUMO

Type 2 diabetes is characterised by failure to control glucose homeostasis, with numerous diabetic complications attributable to the resulting exposure of cells and tissues to chronic elevated concentrations of glucose and fatty acids. This, in part, results from formation of advanced glycation and advanced lipidation end-products that are able to modify protein, lipid, or DNA structure, and disrupt normal cellular function. Herein we used mass spectrometry to identify proteins modified by two such adduction events in serum of individuals with obesity, type 2 diabetes, and gestational diabetes, along with similar analyses of human and mouse skeletal muscle cells and mouse pancreatic islets exposed to glucolipotoxic stress. We also report that carnosine, a histidine containing dipeptide, prevented 65-90% of 4-hydroxynonenal and 3-nitrotyrosine adduction events, and that this in turn preserved mitochondrial function and protected stimulus-secretion coupling in cells exposed to metabolic stress. Carnosine therefore offers significant therapeutic potential against metabolic diseases.


Assuntos
Carnosina , Complicações do Diabetes , Diabetes Mellitus Tipo 2 , Animais , Carnosina/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Produtos Finais de Glicação Avançada/metabolismo , Camundongos , Estresse Oxidativo , Carbonilação Proteica
6.
Mol Metab ; 41: 101047, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32599074

RESUMO

OBJECTIVE: Psoriasis is a chronic inflammatory skin disease that is thought to affect ∼2% of the global population. Psoriasis has been associated with ∼30% increased risk of developing type 2 diabetes (T2D), with numerous studies reporting that psoriasis is an independent risk-factor for T2D, separate from underlying obesity. Separately, studies of skin-specific transgenic mice have reported altered whole-body glucose homeostasis in these models. These studies imply a direct role for skin inflammation and dysfunction in mediating the onset of T2D in psoriasis patients, potentially via the endocrine effects of the skin secretome on key metabolic tissues. We used a combination of in vivo and ex vivo mouse models and ex vivo human imiquimod (IMQ) models to investigate the effects of psoriasis-mediated changes in the skin secretome on whole-body metabolic function. METHODS: To induce psoriatic skin inflammation, mice were topically administered 75 mg of 5% IMQ cream (or Vaseline control) to a shaved dorsal region for 4 consecutive days. On day 5, mice were fasted for glucose and insulin tolerance testing, or sacrificed in the fed state with blood and tissues collected for analysis. To determine effects of the skin secretome, mouse skin was collected at day 5 from IMQ mice and cultured for 24 h. Conditioned media (CM) was collected and used 1:1 with fresh media to treat mouse explant subcutaneous adipose tissue (sAT) and isolated pancreatic islets. For human CM experiments, human skin was exposed to 5% IMQ cream for 20 min, ex vivo, to induce a psoriatic phenotype, then cultured for 24 h. CM was collected, combined 1:1 with fresh media and used to treat human sAT ex vivo. Markers of tissue inflammation and metabolic function were determined by qPCR. Beta cell function in isolated islets was measured by dynamic insulin secretion. Beta-cell proliferation was determined by measurement of Ki67 immunofluorescence histochemistry and BrDU uptake, whilst islet apoptosis was assessed by caspase 3/7 activity. All data is expressed as mean ± SEM. RESULTS: Topical treatment with IMQ induced a psoriatic-like phenotype in mouse skin, evidenced by thickening, erythema and inflammation of the skin. Topical IMQ treatment induced inflammation and signs of metabolic dysfunction in sub-cutaneous and epidydimal adipose tissue, liver, skeletal muscle and gut tissue. However, consistent with islet compensation and a pre-diabetic phenotype, IMQ mice displayed improved glucose tolerance, increased insulin and c-peptide response to glucose, and increased beta cell proliferation. Treatment of sAT with psoriatic mouse or human skin-CM replicated the in vivo phenotype, leading to increased inflammation and metabolic dysfunction in mouse and human sAT. Treatment of pancreatic islets with psoriatic mouse skin-CM induced increases in beta-proliferation and apoptosis, thus partially replicating the in vivo phenotype. CONCLUSIONS: Psoriasis-like skin inflammation induces a pre-diabetic phenotype, characterised by tissue inflammation and markers of metabolic dysfunction, together with islet compensation in mice. The in vivo phenotype is partially replicated by exposure of sAT and pancreatic islets to psoriatic-skin conditioned media. These results support the hypothesis that psoriatic skin inflammation, potentially via the endocrine actions of the skin secretome, may constitute a novel pathophysiological pathway mediating the development of T2D.


Assuntos
Estado Pré-Diabético/etiologia , Estado Pré-Diabético/metabolismo , Psoríase/imunologia , Animais , Proliferação de Células/efeitos dos fármacos , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Imiquimode/metabolismo , Imiquimode/farmacologia , Inflamação/metabolismo , Insulina/metabolismo , Secreção de Insulina/fisiologia , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Psoríase/fisiopatologia , Pele/efeitos dos fármacos , Pele/imunologia , Pele/metabolismo
7.
Biochem Pharmacol ; 171: 113685, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31678493

RESUMO

The worldwide prevalence of diabetes has reached 8.5% among adults, and this is characterised by elevated glucose concentrations and failing insulin secretion. Furthermore, most people with type 2 diabetes are either obese or overweight, with the associated dyslipidaemia contributing to the development of insulin resistance and increased cardiovascular risk. Here we incubated INS-1 pancreatic ß-cells for 72 h in RPMI-1640 media, or media supplemented with 28 mM glucose, 200 µM palmitic acid, and 200 µM oleic acid as a cellular model of diabetic glucolipotoxicity. Illumina HiSeq gene expression analysis showed the trace amine-associated receptor (TAAR) family to be among the most highly downregulated by glucolipotoxicity. Importantly, MetaCore integrated knowledge database, from Clarivate Analytics, indicated potential TAAR impact on insulin secretion through adenylyl cyclase signalling pathways. We therefore investigated the effect of TAAR ligands on cAMP signalling and insulin secretion, and found that only the branch of the TAAR family tree that is activated by isopentylamine, 2-phenylethylamine, p-tyramine, and agmatine significantly increased intracellular cAMP and resulted in increased insulin secretion from INS-1 cells and primary mouse islets under normal conditions. Crucially however, this enhancement was not evident when the receptor family was downregulated by glucolipotoxic conditions. This data indicates that a subset of TAARs are regulators of insulin secretion in pancreatic ß-cells, and that their downregulation contributes to glucolipotoxic inhibition of insulin secretion. As such they may be potential targets for treatment of type 2 diabetes.


Assuntos
Glucose/farmacologia , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Ácido Oleico/farmacologia , Ácido Palmítico/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Linhagem Celular Tumoral , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Resistência à Insulina/genética , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Ligantes , Masculino , Camundongos , Ratos , Receptores Acoplados a Proteínas G/genética
8.
Sci Rep ; 7(1): 13313, 2017 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-29042678

RESUMO

The worldwide prevalence of diabetes has risen to 8.5% among adults, which represents a staggering rise in prevalence from 4.7% in 1980. Whilst some treatments work by increasing insulin secretion, over time their effectiveness decreases. We aim to increase insulin secretion by developing strategies that work through mechanisms independent of current treatment options. Isolated CD1 mouse islets, INS-1 pancreatic ß-cells, or C2C12 mouse myotubes were incubated in standard tissue culture media, or media supplemented with 28 mM glucose, 200 µM palmitic acid, and 200 µM oleic acid as a cellular model of diabetic glucolipotoxicity. Intracellular reactive species content was assayed using 2',7'-dichlorofluorescein diacetate dye, inducible nitric oxide synthase levels determined by Western blot, 3-nitrotyrosine and 4-hydrpxnonenal both assayed by ELISA, insulin secretion quantified using ELISA or radioimmunoassay, and glucose uptake determined through 2-deoxy glucose 6 phosphate luminescence. Our data indicate that carnosine, a histidine containing dipeptide available through the diet, is an effective scavenger of each of the aforementioned reactive species. This results in doubling of insulin secretion from isolated mouse islets or INS-1 ß-cells. Crucially, carnosine also reverses glucolipotoxic inhibition of insulin secretion and enhances glucose uptake into skeletal muscle cells. Thus, carnosine, or non-hydrolysable carnosine analogs, may represent a new class of therapeutic agent to fight type 2 diabetes.


Assuntos
Carnosina/farmacologia , Sequestradores de Radicais Livres/farmacologia , Glucose/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Animais , Linhagem Celular , Radicais Livres/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Camundongos
9.
Diabetes ; 65(5): 1268-82, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26861785

RESUMO

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.


Assuntos
Regulação para Baixo , Ácidos Graxos não Esterificados/metabolismo , Regulação Enzimológica da Expressão Gênica , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Palmitoil-CoA Hidrolase/metabolismo , Animais , Sinalização do Cálcio , Linhagem Celular Tumoral , Células Clonais , Feminino , Intolerância à Glucose/enzimologia , Intolerância à Glucose/metabolismo , Intolerância à Glucose/patologia , Secreção de Insulina , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Especificidade de Órgãos , Palmitoil-CoA Hidrolase/genética , Ratos , Proteínas Recombinantes/metabolismo , Caracteres Sexuais , Técnicas de Cultura de Tecidos , Regulação para Cima
10.
PLoS One ; 11(3): e0149549, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27010458

RESUMO

BACKGROUND: Enteroendocrine L-cells synthesise and release the gut hormone glucagon-like peptide-1 (GLP-1) in response to food transit. Deletion of the tumour suppressor kinase LKB1 from proglucagon-expressing cells leads to the generation of intestinal polyps but no change in circulating GLP-1 levels. Here, we explore the role of the downstream kinase AMP-activated protein kinase (AMPK) in these cells. METHOD: Loss of AMPK from proglucagon-expressing cells was achieved using a preproglucagon promoter-driven Cre (iGluCre) to catalyse recombination of floxed alleles of AMPKα1 and α2. Oral and intraperitoneal glucose tolerance were measured using standard protocols. L-cell mass was measured by immunocytochemistry. Hormone and peptide levels were measured by electrochemical-based luminescence detection or radioimmunoassay. RESULTS: Recombination with iGluCre led to efficient deletion of AMPK from intestinal L- and pancreatic alpha-cells. In contrast to mice rendered null for LKB1 using the same strategy, mice deleted for AMPK displayed an increase (WT: 0.05 ± 0.01, KO: 0.09±0.02%, p<0.01) in L-cell mass and elevated plasma fasting (WT: 5.62 ± 0.800 pg/ml, KO: 14.5 ± 1.870, p<0.01) and fed (WT: 15.7 ± 1.48pg/ml, KO: 22.0 ± 6.62, p<0.01) GLP-1 levels. Oral, but not intraperitoneal, glucose tolerance was significantly improved by AMPK deletion, whilst insulin and glucagon levels were unchanged despite an increase in alpha to beta cell ratio (WT: 0.23 ± 0.02, KO: 0.33 ± 0.03, p<0.01). CONCLUSION: AMPK restricts L-cell growth and GLP-1 secretion to suppress glucose tolerance. Targeted inhibition of AMPK in L-cells may thus provide a new therapeutic strategy in some forms of type 2 diabetes.


Assuntos
Proteínas Quinases Ativadas por AMP/genética , Deleção de Genes , Peptídeo 1 Semelhante ao Glucagon/sangue , Teste de Tolerância a Glucose , Proglucagon/genética , Animais , Feminino , Células Secretoras de Glucagon/citologia , Células Secretoras de Glucagon/metabolismo , Glucose/metabolismo , Insulina/sangue , Mucosa Intestinal/metabolismo , Intestinos/citologia , Masculino , Camundongos , Regiões Promotoras Genéticas
11.
Dis Model Mech ; 7(11): 1275-86, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25190708

RESUMO

Liver kinase B1 (LKB1; also known as STK11) is a serine/threonine kinase and tumour suppressor that is mutated in Peutz-Jeghers syndrome (PJS), a premalignant syndrome associated with the development of gastrointestinal polyps. Proglucagon-expressing enteroendocrine cells are involved in the control of glucose homeostasis and the regulation of appetite through the secretion of gut hormones such as glucagon-like peptide-1 (GLP-1) and peptide tyrosine tyrosine (PYY). To determine the role of LKB1 in these cells, we bred mice bearing floxed alleles of Lkb1 against animals carrying Cre recombinase under proglucagon promoter control. These mice (GluLKB1KO) were viable and displayed near-normal growth rates and glucose homeostasis. However, they developed large polyps at the gastro-duodenal junction, and displayed premature mortality (death from 120 days of age). Histological analysis of the polyps demonstrated that they had a PJS-like appearance with an arborising smooth-muscle core. Circulating GLP-1 levels were normal in GluLKB1KO mice and the polyps expressed low levels of the peptide, similar to levels in the neighbouring duodenum. Lineage tracing using a Rosa26tdRFP transgene revealed, unexpectedly, that enterocytes within the polyps were derived from non-proglucagon-expressing precursors, whereas connective tissue was largely derived from proglucagon-expressing precursors. Developmental studies in wild-type mice suggested that a subpopulation of proglucagon-expressing cells undergo epithelial-mesenchymal transition (EMT) to become smooth-muscle-like cells. Thus, it is likely that polyps in the GluLKB1KO mice developed from a unique population of smooth-muscle-like cells derived from a proglucagon-expressing precursor. The loss of LKB1 within this subpopulation seems to be sufficient to drive tumorigenesis.


Assuntos
Pólipos Intestinais/patologia , Proglucagon/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP , Animais , Transição Epitelial-Mesenquimal , Camundongos , Camundongos Knockout
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