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1.
Diabetologia ; 62(1): 99-111, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30334081

RESUMO

AIMS/HYPOTHESIS: Pancreatic beta cells secrete insulin to maintain glucose homeostasis, and beta cell failure is a hallmark of type 2 diabetes. Glucose triggers insulin secretion in beta cells via oxidative mitochondrial pathways. However, it also feeds mitochondrial anaplerotic pathways, driving citrate export and cytosolic malonyl-CoA production by the acetyl-CoA carboxylase 1 (ACC1) enzyme. This pathway has been proposed as an alternative glucose-sensing mechanism, supported mainly by in vitro data. Here, we sought to address the role of the beta cell ACC1-coupled pathway in insulin secretion and glucose homeostasis in vivo. METHODS: Acaca, encoding ACC1 (the principal ACC isoform in islets), was deleted in beta cells of mice using the Cre/loxP system. Acaca floxed mice were crossed with Ins2cre mice (ßACC1KO; life-long beta cell gene deletion) or Pdx1creER mice (tmx-ßACC1KO; inducible gene deletion in adult beta cells). Beta cell function was assessed using in vivo metabolic physiology and ex vivo islet experiments. Beta cell mass was analysed using histological techniques. RESULTS: ßACC1KO and tmx-ßACC1KO mice were glucose intolerant and had defective insulin secretion in vivo. Isolated islet studies identified impaired insulin secretion from beta cells, independent of changes in the abundance of neutral lipids previously implicated as amplification signals. Pancreatic morphometry unexpectedly revealed reduced beta cell size in ßACC1KO mice but not in tmx-ßACC1KO mice, with decreased levels of proteins involved in the mechanistic target of rapamycin kinase (mTOR)-dependent protein translation pathway underpinning this effect. CONCLUSIONS/INTERPRETATION: Our study demonstrates that the beta cell ACC1-coupled pathway is critical for insulin secretion in vivo and ex vivo and that it is indispensable for glucose homeostasis. We further reveal a role for ACC1 in controlling beta cell growth prior to adulthood.


Assuntos
Acetil-CoA Carboxilase/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Acetil-CoA Carboxilase/genética , Animais , Feminino , Secreção de Insulina/genética , Secreção de Insulina/fisiologia , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Masculino , Camundongos , Camundongos Knockout , Serina-Treonina Quinases TOR/metabolismo
2.
J Biol Chem ; 288(37): 26569-82, 2013 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-23897822

RESUMO

Chronic saturated fatty acid exposure causes ß-cell apoptosis and, thus, contributes to type 2 diabetes. Although endoplasmic reticulum (ER) stress and reduced ER-to-Golgi protein trafficking have been implicated, the exact mechanisms whereby saturated fatty acids trigger ß-cell death remain elusive. Using mass spectroscopic lipidomics and subcellular fractionation, we demonstrate that palmitate pretreatment of MIN6 ß-cells promoted ER remodeling of both phospholipids and sphingolipids, but only the latter was causally linked to lipotoxic ER stress. Thus, overexpression of glucosylceramide synthase, previously shown to protect against defective protein trafficking and ER stress, partially reversed lipotoxic reductions in ER sphingomyelin (SM) content and aggregation of ER lipid rafts, as visualized using Erlin1-GFP. Using both lipidomics and a sterol response element reporter assay, we confirmed that free cholesterol in the ER was also reciprocally modulated by chronic palmitate and glucosylceramide synthase overexpression. This is consistent with the known coregulation and association of SM and free cholesterol in lipid rafts. Inhibition of SM hydrolysis partially protected against ATF4/C/EBP homology protein induction because of palmitate. Our results suggest that loss of SM in the ER is a key event for initiating ß-cell lipotoxicity, which leads to disruption of ER lipid rafts, perturbation of protein trafficking, and initiation of ER stress.


Assuntos
Retículo Endoplasmático/metabolismo , Células Secretoras de Insulina/citologia , Lipídeos/química , Microdomínios da Membrana/química , Animais , Apoptose , Linhagem Celular , Ceramidas/química , Colesterol/química , Diabetes Mellitus Tipo 2/metabolismo , Ácidos Graxos/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ácido Palmítico/química , Transporte Proteico , Esfingomielinas/química , Frações Subcelulares/metabolismo
3.
Sci Adv ; 9(37): eadh0831, 2023 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-37703359

RESUMO

The incidence of hepatocellular carcinoma (HCC) is rapidly rising largely because of increased obesity leading to nonalcoholic steatohepatitis (NASH), a known HCC risk factor. There are no approved treatments to treat NASH. Here, we first used single-nucleus RNA sequencing to characterize a mouse model that mimics human NASH-driven HCC, the MUP-uPA mouse fed a high-fat diet. Activation of endoplasmic reticulum (ER) stress and inflammation was observed in a subset of hepatocytes that was enriched in mice that progress to HCC. We next treated MUP-uPA mice with the ER stress inhibitor BGP-15 and soluble gp130Fc, a drug that blocks inflammation by preventing interleukin-6 trans-signaling. Both drugs have progressed to phase 2/3 human clinical trials for other indications. We show that this combined therapy reversed NASH and reduced NASH-driven HCC. Our data suggest that these drugs could provide a potential therapy for NASH progression to HCC.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Hepatopatia Gordurosa não Alcoólica , Humanos , Animais , Camundongos , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/prevenção & controle , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/prevenção & controle , Hepatócitos , Inflamação/tratamento farmacológico
4.
Cell Genom ; 3(5): 100301, 2023 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-37228755

RESUMO

Current approaches to staging chronic liver diseases have limited utility for predicting liver cancer risk. Here, we employed single-nucleus RNA sequencing (snRNA-seq) to characterize the cellular microenvironment of healthy and pre-malignant livers using two distinct mouse models. Downstream analyses unraveled a previously uncharacterized disease-associated hepatocyte (daHep) transcriptional state. These cells were absent in healthy livers but increasingly prevalent as chronic liver disease progressed. Copy number variation (CNV) analysis of microdissected tissue demonstrated that daHep-enriched regions are riddled with structural variants, suggesting these cells represent a pre-malignant intermediary. Integrated analysis of three recent human snRNA-seq datasets confirmed the presence of a similar phenotype in human chronic liver disease and further supported its enhanced mutational burden. Importantly, we show that high daHep levels precede carcinogenesis and predict a higher risk of hepatocellular carcinoma development. These findings may change the way chronic liver disease patients are staged, surveilled, and risk stratified.

5.
Biochem J ; 435(1): 267-76, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21265737

RESUMO

Saturated fatty acids promote lipotoxic ER (endoplasmic reticulum) stress in pancreatic ß-cells in association with Type 2 diabetes. To address the underlying mechanisms we employed MS in a comprehensive lipidomic screen of MIN6 ß-cells treated for 48 h with palmitate. Both the overall mass and the degree of saturation of major neutral lipids and phospholipids were only modestly increased by palmitate. The mass of GlcCer (glucosylceramide) was augmented by 70% under these conditions, without any significant alteration in the amounts of either ceramide or sphingomyelin. However, flux into ceramide (measured by [3H]serine incorporation) was augmented by chronic palmitate, and inhibition of ceramide synthesis decreased both ER stress and apoptosis. ER-to-Golgi protein trafficking was also reduced by palmitate pre-treatment, but was overcome by overexpression of GlcCer synthase. This was accompanied by increased conversion of ceramide into GlcCer, and reduced ER stress and apoptosis, but no change in phospholipid desaturation. Sphingolipid alterations due to palmitate were not secondary to ER stress since they were neither reproduced by pharmacological ER stressors nor overcome using the chemical chaperone phenylbutyric acid. In conclusion, alterations in sphingolipid, rather than phospholipid, metabolism are more likely to be implicated in the defective protein trafficking and enhanced ER stress and apoptosis of lipotoxic ß-cells.


Assuntos
Retículo Endoplasmático/metabolismo , Células Secretoras de Insulina/metabolismo , Ácido Palmítico/metabolismo , Esfingolipídeos/metabolismo , Estresse Fisiológico , Animais , Apoptose , Biomarcadores/metabolismo , Linhagem Celular , Retículo Endoplasmático/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Glucosilceramidas/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Metabolismo dos Lipídeos , Metabolômica/métodos , Camundongos , Ácido Palmítico/efeitos adversos , Fenilbutiratos/farmacologia , Biossíntese de Proteínas , Transporte Proteico , Serina C-Palmitoiltransferase/antagonistas & inibidores , Estresse Fisiológico/efeitos dos fármacos , Tapsigargina/toxicidade , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Tunicamicina/toxicidade
6.
Cancers (Basel) ; 12(7)2020 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-32605253

RESUMO

Obesity is recognised as a risk factor for many types of cancers, in particular hepatocellular carcinoma (HCC). A critical factor in the development of HCC from non-alcoholic fatty liver disease (NAFLD) is the presence of non-alcoholic steatohepatitis (NASH). Therapies aimed at NASH to reduce the risk of HCC are sparse and largely unsuccessful. Lifestyle modifications such as diet and regular exercise have poor adherence. Moreover, current pharmacological treatments such as pioglitazone and vitamin E have limited effects on fibrosis, a key risk factor in HCC progression. As NAFLD is becoming more prevalent in developed countries due to rising rates of obesity, a need for directed treatment is imperative. Numerous novel therapies including PPAR agonists, anti-fibrotic therapies and agents targeting inflammation, oxidative stress and the gut-liver axis are currently in development, with the aim of targeting key processes in the progression of NASH and HCC. Here, we critically evaluate literature on the aetiology of NAFLD-related HCC, and explore the potential treatment options for NASH and HCC.

7.
Cancer Cell ; 37(3): 354-370.e7, 2020 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-32183951

RESUMO

Immunotherapy has emerged as a powerful new chapter in the fight against cancer. However, it has yet to reach its full potential due in part to the complexity of the cancer immune response. We demonstrate that tumor-targeting EDV nanocells function as an immunotherapeutic by delivering a cytotoxin in conjunction with activation of the immune system. These nanocells polarize M1 macrophages and activate NK cells concurrently producing a Th1 cytokine response resulting in potent antitumor function. Dendritic cell maturation and antigen presentation follows, which generates tumor-specific CD8+ T cells, conferring prolonged tumor remission. The combination of cytotoxin delivery and activation of innate and adaptive antitumor immune responses results in a potent cyto-immunotherapeutic with potential in clinical oncology.


Assuntos
Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Imunidade Inata/efeitos dos fármacos , Salmonella typhimurium/citologia , Adulto , Idoso , Animais , Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Linhagem Celular , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/fisiologia , Doxorrubicina/administração & dosagem , Doxorrubicina/análogos & derivados , Receptores ErbB/administração & dosagem , Receptores ErbB/metabolismo , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Humanos , Imunoterapia/métodos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Nanoestruturas/química , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia
8.
Mol Metab ; 5(6): 404-414, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27257600

RESUMO

OBJECTIVE: Glucose promotes lipid remodelling in pancreatic ß-cells, and this is thought to contribute to the regulation of insulin secretion, but the metabolic pathways and potential signalling intermediates have not been fully elaborated. METHODS: Using mass spectrometry (MS) we quantified changes in approximately 300 lipid metabolites in MIN6 ß-cells and isolated mouse islets following 1 h stimulation with glucose. Flux through sphingolipid pathways was also assessed in (3)H-sphinganine-labelled cells using TLC. RESULTS: Glucose specifically activates the conversion of triacylglycerol (TAG) to diacylglycerol (DAG). This leads indirectly to the formation of 18:1 monoacylglycerol (MAG), via degradation of saturated/monounsaturated DAG species, such as 16:0_18:1 DAG, which are the most abundant, immediate products of glucose-stimulated TAG hydrolysis. However, 16:0-containing, di-saturated DAG species are a better direct marker of TAG hydrolysis since, unlike the 18:1-containing DAGs, they are predominately formed via this route. Using multiple reaction monitoring, we confirmed that in islets under basal conditions, 18:1 MAG is the most abundant species. We further demonstrated a novel site of glucose to enhance the conversion of ceramide to sphingomyelin (SM) and galactosylceramide (GalCer). Flux and product:precursor analyses suggest regulation of the enzyme SM synthase, which would constitute a separate mechanism for localized generation of DAG in response to glucose. Phosphatidylcholine (PC) plasmalogen (P) species, specifically those containing 20:4, 22:5 and 22:6 side chains, were also diminished in the presence of glucose, whereas the more abundant phosphatidylethanolamine plasmalogens were unchanged. CONCLUSION: Our results highlight 18:1 MAG, GalCer, PC(P) and DAG/SM as potential contributors to metabolic stimulus-secretion coupling.

9.
Trends Endocrinol Metab ; 25(8): 389-98, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24656915

RESUMO

Failure of the unfolded protein response (UPR) to maintain optimal folding of pro-insulin in the endoplasmic reticulum (ER) leads to unresolved ER stress and ß cell death. This contributes not only to some rare forms of diabetes, but also to type 2 diabetes mellitus (T2DM). Many key findings, elaborated over the past decade, are based on the lipotoxicity model, entailing chronic exposure of ß cells to elevated levels of fatty acids (FAs). Here, we update recent progress on how FAs initiate ER stress, particularly via disruption of protein trafficking, and how this leads to apoptosis. We also highlight differences in how ß cells are impacted by the classic UPR, versus the more selective UPR that arises as part of a broader response to lipotoxicity.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Células Secretoras de Insulina/metabolismo , Animais , Apoptose/fisiologia , Retículo Endoplasmático/metabolismo , Resposta a Proteínas não Dobradas/fisiologia
10.
Islets ; 4(3): 177-87, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22847494

RESUMO

Recent technical advances have re-invigorated the study of sphingolipid metabolism in general, and helped to highlight the varied and important roles that sphingolipids play in pancreatic ß-cells. Sphingolipid metabolites such as ceramide, glycosphingolipids, sphingosine 1-phosphate and gangliosides modulate many ß-cell signaling pathways and processes implicated in ß-cell diabetic disease such as apoptosis, ß-cell cytokine secretion, ER-to-golgi vesicular trafficking, islet autoimmunity and insulin gene expression. They are particularly relevant to lipotoxicity. Moreover, the de novo synthesis of sphingolipids occurs on many subcellular membranes, in parallel to secretory vesicle formation, traffic and granule maturation events. Indeed, the composition of the plasma membrane, determined by the activity of neutral sphingomyelinases, affects ß-cell excitability and potentially insulin exocytosis while another glycosphingolipid, sulfatide, determines the stability of insulin crystals in granules. Most importantly, sphingolipid metabolism on internal membranes is also strongly implicated in regulating ß-cell apoptosis.


Assuntos
Ceramidas/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Esfingolipídeos/metabolismo , Animais , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 2/patologia , Humanos , Transdução de Sinais
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