RESUMO
AIM: Acyl-coenzyme A dehydrogenase family member 10 (ACAD10) is a mitochondrial protein purported to be involved in the fatty acid oxidation pathway. Metformin is the most prescribed therapy for type 2 diabetes; however, its precise mechanisms of action(s) are still being uncovered. Upregulation of ACAD10 is a requirement for metformin's ability to inhibit growth in cancer cells and extend lifespan in Caenorhabditis elegans. However, it is unknown whether ACAD10 plays a role in metformin's metabolic actions. MATERIALS AND METHODS: We assessed the role for ACAD10 on whole-body metabolism and metformin action by generating ACAD10KO mice on a C57BL/6J background via CRISPR-Cas9 technology. In-depth metabolic phenotyping was conducted in both sexes on a normal chow and high fat-high sucrose diet. RESULTS: Compared with wildtype mice, we detected no difference in body composition, energy expenditure or glucose tolerance in male or female ACAD10KO mice, on a chow diet or high-fat, high-sucrose diet (p ≥ .05). Hepatic mitochondrial function and insulin signalling was not different between genotypes under basal or insulin-stimulated conditions (p ≥ .05). Glucose excursions following acute administration of metformin before a glucose tolerance test were not different between genotypes nor was body composition or energy expenditure altered after 4 weeks of daily metformin treatment (p ≥ .05). Despite the lack of a metabolic phenotype, liver lipidomic analysis suggests ACAD10 depletion influences the abundance of specific ceramide species containing very long chain fatty acids, while metformin treatment altered clusters of cholesterol ester, plasmalogen, phosphatidylcholine and ceramide species. CONCLUSIONS: Loss of ACAD10 does not alter whole-body metabolism or impact the acute or chronic metabolic actions of metformin in this model.
Assuntos
Diabetes Mellitus Tipo 2 , Metformina , Masculino , Feminino , Camundongos , Animais , Diabetes Mellitus Tipo 2/metabolismo , Camundongos Endogâmicos C57BL , Metformina/farmacologia , Glucose/metabolismo , Insulina , Ceramidas , Sacarose , Dieta Hiperlipídica/efeitos adversosRESUMO
Lipotoxicity, the accumulation of lipids in non-adipose tissues, alters the metabolic transcriptome and mitochondrial metabolism in skeletal muscle. The mechanisms involved remain poorly understood. Here we show that lipotoxicity increased histone deacetylase 4 (HDAC4) and histone deacetylase 5 (HDAC5), which reduced the expression of metabolic genes and oxidative metabolism in skeletal muscle, resulting in increased non-oxidative glucose metabolism. This metabolic reprogramming was also associated with impaired apoptosis and ferroptosis responses, and preserved muscle cell viability in response to lipotoxicity. Mechanistically, increased HDAC4 and 5 decreased acetylation of p53 at K120, a modification required for transcriptional activation of apoptosis. Redox drivers of ferroptosis derived from oxidative metabolism were also reduced. The relevance of this pathway was demonstrated by overexpression of loss-of-function HDAC4 and HDAC5 mutants in skeletal muscle of obese db/db mice, which enhanced oxidative metabolic capacity, increased apoptosis and ferroptosis and reduced muscle mass. This study identifies HDAC4 and HDAC5 as repressors of skeletal muscle oxidative metabolism, which is linked to inhibition of cell death pathways and preservation of muscle integrity in response to lipotoxicity.
Assuntos
Histona Desacetilases , Células Musculares , Camundongos , Animais , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Células Musculares/metabolismo , Músculo Esquelético/metabolismo , Processamento de Proteína Pós-Traducional , Morte CelularRESUMO
Type 2 diabetes mellitus (T2DM), a condition characterised by insulin resistance (IR) and skeletal muscle mitochondrial abnormalities, is a leading cause of death in developed societies. Much work has postulated that improving pathways linked to mitochondrial health, including autophagy, may be a potential avenue to prevent or treat T2DM. Given the recent data indicating a role for tripartite motif-containing 28 (TRIM28) in autophagy and mitochondrial pathways, we investigated whether muscle-specific deletion of TRIM28 might impact on obesity, glucose tolerance, and IR in mice. We studied two different muscle-specific (MCK-cre and ACTA1-cre-ERT2) TRIM28 knockout models, which were phenotyped during and after being fed a chow or high-fat diet (HFD). Whilst muscle-specific deletion of TRIM28 in both models demonstrated alterations in markers of mitochondrial activity and autophagy in skeletal muscle, we did not observe major impacts on the majority of metabolic measures in these mice. Specifically, we demonstrate that deletion of TRIM28 in skeletal muscle of mice during (MCK-cre) or post-development (ACTA1-cre-ERT2) does not prevent HFD-induced obesity or glucose intolerance. These findings are consistent with those reported previously in relation to autophagy and mitochondria in other cell types, and thus warrant further study into the biological role TRIM28 has in relation to mitochondrial function.
Assuntos
Diabetes Mellitus Tipo 2 , Intolerância à Glucose , Resistência à Insulina , Camundongos , Animais , Diabetes Mellitus Tipo 2/metabolismo , Resistência à Insulina/genética , Músculo Esquelético/metabolismo , Intolerância à Glucose/metabolismo , Obesidade/metabolismo , Dieta Hiperlipídica/efeitos adversos , Camundongos Endogâmicos C57BL , Mitocôndrias Musculares/metabolismo , Proteína 28 com Motivo Tripartido/metabolismoRESUMO
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 farmacológicoRESUMO
AIMS: The glucose-driven enzymatic modification of myocardial proteins by the sugar moiety, ß-N-acetylglucosamine (O-GlcNAc), is increased in pre-clinical models of diabetes, implicating protein O-GlcNAc modification in diabetes-induced heart failure. Our aim was to specifically examine cardiac manipulation of the two regulatory enzymes of this process on the cardiac phenotype, in the presence and absence of diabetes, utilising cardiac-targeted recombinant-adeno-associated viral-vector-6 (rAAV6)-mediated gene delivery. METHODS AND RESULTS: In human myocardium, total protein O-GlcNAc modification was elevated in diabetic relative to non-diabetic patients, and correlated with left ventricular (LV) dysfunction. The impact of rAAV6-delivered O-GlcNAc transferase (rAAV6-OGT, facilitating protein O-GlcNAcylation), O-GlcNAcase (rAAV6-OGA, facilitating de-O-GlcNAcylation), and empty vector (null) were determined in non-diabetic and diabetic mice. In non-diabetic mice, rAAV6-OGT was sufficient to impair LV diastolic function and induce maladaptive cardiac remodelling, including cardiac fibrosis and increased Myh-7 and Nppa pro-hypertrophic gene expression, recapitulating characteristics of diabetic cardiomyopathy. In contrast, rAAV6-OGA (but not rAAV6-OGT) rescued LV diastolic function and adverse cardiac remodelling in diabetic mice. Molecular insights implicated impaired cardiac PI3K(p110α)-Akt signalling as a potential contributing mechanism to the detrimental consequences of rAAV6-OGT in vivo. In contrast, rAAV6-OGA preserved PI3K(p110α)-Akt signalling in diabetic mouse myocardium in vivo and prevented high glucose-induced impairments in mitochondrial respiration in human cardiomyocytes in vitro. CONCLUSION: Maladaptive protein O-GlcNAc modification is evident in human diabetic myocardium, and is a critical regulator of the diabetic heart phenotype. Selective targeting of cardiac protein O-GlcNAcylation to restore physiological O-GlcNAc balance may represent a novel therapeutic approach for diabetes-induced heart failure.
Assuntos
Antígenos de Neoplasias/metabolismo , Cardiomiopatias Diabéticas/enzimologia , Histona Acetiltransferases/metabolismo , Hialuronoglucosaminidase/metabolismo , Miócitos Cardíacos/enzimologia , N-Acetilglucosaminiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Disfunção Ventricular Esquerda/enzimologia , Função Ventricular Esquerda , Remodelação Ventricular , Idoso , Animais , Antígenos de Neoplasias/genética , Linhagem Celular , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Cardiomiopatias Diabéticas/genética , Cardiomiopatias Diabéticas/patologia , Cardiomiopatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Feminino , Fibrose , Regulação da Expressão Gênica , Glicosilação , Histona Acetiltransferases/genética , Humanos , Hialuronoglucosaminidase/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Miócitos Cardíacos/patologia , N-Acetilglucosaminiltransferases/genética , Fenótipo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/fisiopatologiaRESUMO
Identifying the fundamental molecular factors that drive weight gain even in the absence of hypercaloric food intake, is crucial to enable development of novel treatments for the global pandemic of obesity. Here we investigated both adipose tissue-specific and systemic events that underlie the physiological weight gain occurring during early adulthood in mice fed a normocaloric diet. In addition, we used three different genetic models to identify molecular factors that promote physiological weight gain during normocaloric and hypercaloric diets. We demonstrated that normal physiological weight gain was accompanied by an increase in adipose tissue mass and the presence of cellular and metabolic signatures typically found during obesity, including adipocyte hypertrophy, macrophage recruitment into visceral fat and perturbed glucose metabolism. At the molecular level, this was associated with an increase in adipose tissue tryptophan hydroxylase 1 (Tph1) transcripts, the key enzyme responsible for the synthesis of peripheral serotonin. Genetic inactivation of Tph1 was sufficient to limit adipose tissue expansion and associated metabolic alterations. Mechanistically, we discovered that Tph1 inactivation resulted in down-regulation of cyclin-dependent kinase inhibitor p21Waf1/Cip1 expression. Single or double ablation of Tph1 and p21 were equally effective in preventing adipocyte expansion and systemic perturbation of glucose metabolism, upon both normocaloric and hypercaloric diets. Our results suggest that serotonin and p21 act as a central molecular determinant of weight gain and associated metabolic alterations, and highlights the potential of targeting these molecules as a pharmacologic approach to prevent the development of obesity.
Assuntos
Tecido Adiposo/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dieta Saudável/métodos , Deleção de Genes , Obesidade/metabolismo , Serotonina/biossíntese , Transdução de Sinais/genética , Adipócitos/patologia , Animais , Tamanho Celular , Inibidor de Quinase Dependente de Ciclina p21/genética , Dieta Hiperlipídica , Glucose/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/genética , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/metabolismo , Aumento de Peso/genéticaRESUMO
OBJECTIVE: CRISPR/Cas9 technology has revolutionized gene editing and fast tracked our capacity to manipulate genes of interest for the benefit of both research and therapeutic applications. Whilst many advances have, and continue to be made in this area, perhaps the most utilized technology to date has been the generation of knockout cells, tissues and animals. The advantages of this technology are many fold, however some questions still remain regarding the effects that long term expression of foreign proteins such as Cas9, have on mammalian cell function. Several studies have proposed that chronic overexpression of Cas9, with or without its accompanying guide RNAs, may have deleterious effects on cell function and health. This is of particular concern when applying this technology in vivo, where chronic expression of Cas9 in tissues of interest may promote disease-like phenotypes and thus confound the investigation of the effects of the gene of interest. Although these concerns remain valid, no study to our knowledge has yet to demonstrate this directly. METHODS: In this study we used the lox-stop-lox (LSL) spCas9 ROSA26 transgenic (Tg) mouse line to generate four tissue-specific Cas9-Tg models that express Cas9 in the heart, liver, skeletal muscle or adipose tissue. We performed comprehensive phenotyping of these mice up to 20-weeks of age and subsequently performed molecular analysis of their organs. RESULTS: We demonstrate that Cas9 expression in these tissues had no detrimental effect on whole body health of the animals, nor did it induce any tissue-specific effects on whole body energy metabolism, liver health, inflammation, fibrosis, heart function or muscle mass. CONCLUSIONS: Our data suggests that these models are suitable for studying the tissue specific effects of gene deletion using the LSL-Cas9-Tg model, and that phenotypes observed utilizing these models can be confidently interpreted as being gene specific, and not confounded by the chronic overexpression of Cas9.
Assuntos
Proteína 9 Associada à CRISPR/genética , Animais , Sistemas CRISPR-Cas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , FenótipoRESUMO
BACKGROUND: Diabetes mellitus is associated with inadequate delivery of oxygen to tissues. Cellular hypoxia is associated with mitochondrial dysfunction which increases oxidative stress and hyperglycaemia. Hyperbaric oxygenation therapy, which was shown to improve insulin sensitivity, is impractical for regular use. We evaluated the effects of water which is stably-enriched with oxygen (ELO water) to increase arterial blood oxygen levels, on mitochondrial function in the presence of normal- or high-glucose environments, and as glucose-lowering therapy in humans. METHODS: We compared arterial blood oxygen levels in Sprague-Dawley rats after 7 days of ad libitum ELO or tap water consumption. Mitochondrial stress testing, and flow cytometry analysis of mitochondrial mass and membrane potential, were performed on human HepG2 cells cultured in four Dulbecco's Modified Eagle Medium media, made with ELO water or regular (control) water, at normal (5.5 mM) or high (25 mM) glucose concentrations. We also randomized 150 adults with type 2 diabetes (mean age 53 years, glycated haemoglobin HbA1c 8.9% [74 mmol/mol], average duration of diabetes 12 years) to drink 1.5 litres daily of bottled ELO water or drinking water. RESULTS: ELO water raised arterial oxygen tension pO2 significantly (335 ± 26 vs. 188 ± 18 mmHg, p = 0.006) compared with tap water. In cells cultured in control water, mitochondrial mass and membrane potential were both significantly lower at 25 mM glucose compared with 5.5 mM glucose; in contrast, mitochondrial mass and membrane potential did not differ significantly at normal or high glucose concentrations in cells cultured in ELO water. The high-glucose environment induced a greater mitochondrial proton leak in cells cultured in ELO water compared to cells cultured in control medium at similar glucose concentration. In type 2 diabetic adults, HbA1c decreased significantly (p = 0.002) by 0.3 ± 0.7% (4 ± 8 mmol/mol), with ELO water after 12 weeks of treatment but was unchanged with placebo. CONCLUSIONS: ELO water raises arterial blood oxygen levels, appears to have a protective effect on hyperglycaemia-induced reduction in mitochondrial mass and mitochondrial dysfunction, and may be effective adjuvant therapy for type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2 , Oxigênio , Animais , Hemoglobinas Glicadas , Hipoglicemiantes , Insulina , Ratos , Ratos Sprague-Dawley , ÁguaRESUMO
The effective storage of lipids in white adipose tissue (WAT) critically impacts whole body energy homeostasis. Many genes have been implicated in WAT lipid metabolism, including tripartite motif containing 28 (Trim28), a gene proposed to primarily influence adiposity via epigenetic mechanisms in embryonic development. However, in the current study we demonstrate that mice with deletion of Trim28 specifically in committed adipocytes, also develop obesity similar to global Trim28 deletion models, highlighting a post-developmental role for Trim28. These effects were exacerbated in female mice, contributing to the growing notion that Trim28 is a sex-specific regulator of obesity. Mechanistically, this phenotype involves alterations in lipolysis and triglyceride metabolism, explained in part by loss of Klf14 expression, a gene previously demonstrated to modulate adipocyte size and body composition in a sex-specific manner. Thus, these findings provide evidence that Trim28 is a bona fide, sex specific regulator of post-developmental adiposity and WAT function.
Assuntos
Adipócitos/metabolismo , Deleção de Genes , Glucose/metabolismo , Obesidade/patologia , Proteína 28 com Motivo Tripartido/genética , Células 3T3-L1 , Tecido Adiposo Branco/metabolismo , Adiposidade , Animais , Peso Corporal , Dieta , Dieta Hiperlipídica , Metabolismo Energético , Feminino , Redes Reguladoras de Genes , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/genética , Fenótipo , Triglicerídeos/metabolismo , Proteína 28 com Motivo Tripartido/deficiênciaRESUMO
This study analyzed the effects of 24 h of cold stress (22°C or 5°C vs. mice maintained at 30 °C) on the plasma, brown adipose tissue (BAT), subcutaneous (SubQ) and epididymal (Epi) white adipose tissue (WAT), liver, and skeletal muscle lipidome of mice. Using mass spectrometry-lipidomics, 624 lipid species were detected, of which 239 were significantly altered in plasma, 134 in BAT, and 51 in the liver. In plasma, acylcarnitines and free fatty acids were markedly increased at 5°C. Plasma triacylglycerols (TGs) were reduced at 22°C and 5°C. We also identified ether lipids as a novel, cold-induced lipid class. In BAT, TGs were the principal lipid class affected by cold stress, being significantly reduced at both 22°C and 5°C. Interestingly, although BAT TG species were uniformly affected at 5°C, at 22°C we observed species-dependent effects, with TGs containing longer and more unsaturated fatty acids particularly sensitive to the effects of cold. In the liver, TGs were the most markedly affected lipid class, increasing in abundance at 5 °C. TGs containing longer and more unsaturated fatty acids accumulated to a greater degree. Our work demonstrates the following: 1) acute exposure to moderate (22°C) cold stress alters the plasma and BAT lipidome; although this effect is markedly less pronounced than at 5°C. 2) Cold stress at 5°C dramatically alters the plasma lipidome, with ether lipids identified as a novel lipid class altered by cold exposure. 3) Cold-induced alterations in liver and BAT TG levels are not uniform, with changes being influenced by acyl chain composition.
Assuntos
Temperatura Baixa/efeitos adversos , Metabolismo dos Lipídeos/fisiologia , Lipidômica/métodos , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Composição Corporal , Epididimo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Estresse FisiológicoRESUMO
Benign hepatosteatosis, affected by lipid uptake, de novo lipogenesis and fatty acid (FA) oxidation, progresses to non-alcoholic steatohepatitis (NASH) on stress and inflammation. A key macronutrient proposed to increase hepatosteatosis and NASH risk is fructose. Excessive intake of fructose causes intestinal-barrier deterioration and endotoxaemia. However, how fructose triggers these alterations and their roles in hepatosteatosis and NASH pathogenesis remain unknown. Here we show, using mice, that microbiota-derived Toll-like receptor (TLR) agonists promote hepatosteatosis without affecting fructose-1-phosphate (F1P) and cytosolic acetyl-CoA. Activation of mucosal-regenerative gp130 signalling, administration of the YAP-induced matricellular protein CCN1 or expression of the antimicrobial peptide Reg3b (beta) peptide counteract fructose-induced barrier deterioration, which depends on endoplasmic-reticulum stress and subsequent endotoxaemia. Endotoxin engages TLR4 to trigger TNF production by liver macrophages, thereby inducing lipogenic enzymes that convert F1P and acetyl-CoA to FA in both mouse and human hepatocytes.
Assuntos
Frutose/farmacologia , Inflamação/metabolismo , Lipogênese/efeitos dos fármacos , Acetilcoenzima A/farmacologia , Animais , Endotoxemia/sangue , Feminino , Frutosefosfatos/farmacologia , Microbioma Gastrointestinal , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Intestinos/efeitos dos fármacos , Lipidômica , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismo , Regeneração/efeitos dos fármacos , Receptores Toll-Like/agonistasRESUMO
FOXP3+ regulatory T (Treg) cells are essential for maintaining immunological tolerance. Given their importance in immune-related diseases, cancer and obesity, there is increasing interest in targeting the Treg cell compartment therapeutically. New pharmacological inhibitors that specifically target the prosurvival protein MCL-1 may provide this opportunity, as Treg cells are particularly reliant upon this protein. However, there are two distinct isoforms of MCL-1; one located at the outer mitochondrial membrane (OMM) that is required to antagonize apoptosis, and another at the inner mitochondrial membrane (IMM) that is reported to maintain IMM structure and metabolism via ATP production during oxidative phosphorylation. We set out to elucidate the relative importance of these distinct biological functions of MCL-1 in Treg cells to assess whether MCL-1 inhibition might impact upon the metabolism of cells able to resist apoptosis. Conditional deletion of Mcl1 in FOXP3+ Treg cells resulted in a lethal multiorgan autoimmunity due to the depletion of the Treg cell compartment. This striking phenotype was completely rescued by concomitant deletion of the apoptotic effector proteins BAK and BAX, indicating that apoptosis plays a pivotal role in the homeostasis of Treg cells. Notably, MCL-1-deficient Treg cells rescued from apoptosis displayed normal metabolic capacity. Moreover, pharmacological inhibition of MCL-1 in Treg cells resistant to apoptosis did not perturb their metabolic function. We conclude that Treg cells require MCL-1 only to antagonize apoptosis and not for metabolism. Therefore, MCL-1 inhibition could be used to manipulate Treg cell survival for clinical benefit without affecting the metabolic fitness of cells resisting apoptosis.
Assuntos
Apoptose/imunologia , Fatores de Transcrição Forkhead/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Apoptose/genética , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Feminino , Fatores de Transcrição Forkhead/genética , Deleção de Genes , Homeostase/imunologia , Tolerância Imunológica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Transdução de SinaisRESUMO
Studies suggest the gut microbiota contributes to the development of obesity and metabolic syndrome. Exercise alters microbiota composition and diversity and is protective of these maladies. We tested whether the protective metabolic effects of exercise are mediated through fecal components through assessment of body composition and metabolism in recipients of fecal microbiota transplantation (FMT) from exercise-trained (ET) mice fed normal or high-energy diets. Donor C57BL/6J mice were fed a chow or high-fat, high-sucrose diet (HFHS) for 4 wk to induce obesity and glucose intolerance. Mice were divided into sedentary (Sed) or ET groups (6 wk treadmill-based ET) while maintaining their diets, resulting in four donor groups: chow sedentary (NC-Sed) or ET (NC-ET) and HFHS sedentary (HFHS-Sed) or ET (HFHS-ET). Chow-fed recipient mice were gavaged with feces from the respective donor groups weekly, creating four groups (NC-Sed-R, NC-ET-R, HFHS-Sed-R, HFHS-ET-R), and body composition and metabolism were assessed. The HFHS diet led to glucose intolerance and obesity in the donors, whereas exercise training (ET) restrained adiposity and improved glucose tolerance. No donor group FMT altered recipient body composition. Despite unaltered adiposity, glucose levels were disrupted when challenged in mice receiving feces from HFHS-fed donors, irrespective of donor-ET status, with a decrease in insulin-stimulated glucose clearance into white adipose tissue and large intestine and specific changes in the recipient's microbiota composition observed. FMT can transmit HFHS-induced disrupted glucose metabolism to recipient mice independently of any change in adiposity. However, the protective metabolic effect of ET on glucose metabolism is not mediated through fecal factors.
Assuntos
Dieta Hiperlipídica , Sacarose Alimentar , Transplante de Microbiota Fecal , Intolerância à Glucose/microbiologia , Obesidade/microbiologia , Condicionamento Físico Animal , Comportamento Sedentário , Adiposidade , Animais , Microbioma Gastrointestinal , Glucose/metabolismo , Intolerância à Glucose/metabolismo , Masculino , Camundongos , Obesidade/metabolismo , Distribuição AleatóriaRESUMO
AIMS/HYPOTHESIS: Compared with the general population, individuals with diabetes have a higher risk of developing severe acute pancreatitis, a highly debilitating and potentially lethal inflammation of the exocrine pancreas. In this study, we investigated whether 1-deoxysphingolipids, atypical lipids that increase in the circulation following the development of diabetes, exacerbate the severity of pancreatitis in a diabetic setting. METHODS: We analysed whether administration of an L-serine-enriched diet to mouse models of diabetes, an established method for decreasing the synthesis of 1-deoxysphingolipids in vivo, reduced the severity of acute pancreatitis. Furthermore, we elucidated the molecular mechanisms underlying the lipotoxicity exerted by 1-deoxysphingolipids towards rodent pancreatic acinar cells in vitro. RESULTS: We demonstrated that L-serine supplementation reduced the damage of acinar tissue resulting from the induction of pancreatitis in diabetic mice (average histological damage score: 1.5 in L-serine-treated mice vs 2.7 in the control group). At the cellular level, we showed that L-serine decreased the production of reactive oxygen species, endoplasmic reticulum stress and cellular apoptosis in acinar tissue. Importantly, these parameters, together with DNA damage, were triggered in acinar cells upon treatment with 1-deoxysphingolipids in vitro, suggesting that these lipids are cytotoxic towards pancreatic acinar cells in a cell-autonomous manner. In search of the initiating events of the observed cytotoxicity, we discovered that 1-deoxysphingolipids induced early mitochondrial dysfunction in acinar cells, characterised by ultrastructural alterations, impaired oxygen consumption rate and reduced ATP synthesis. CONCLUSIONS/INTERPRETATION: Our results suggest that 1-deoxysphingolipids directly damage the functionality of pancreatic acinar cells and highlight that an L-serine-enriched diet may be used as a promising prophylactic intervention to reduce the severity of pancreatitis in the context of diabetes.
Assuntos
Células Acinares/efeitos dos fármacos , Diabetes Mellitus Experimental/metabolismo , Pâncreas/efeitos dos fármacos , Pancreatite/metabolismo , Serina/farmacologia , Células Acinares/metabolismo , Células Acinares/ultraestrutura , Animais , Apoptose/efeitos dos fármacos , Ceruletídeo/toxicidade , Dano ao DNA/efeitos dos fármacos , Diabetes Mellitus Experimental/complicações , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Técnicas In Vitro , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Pâncreas/citologia , Pancreatite/etiologia , Espécies Reativas de Oxigênio/metabolismo , Índice de Gravidade de Doença , Esfingolipídeos/metabolismo , Esfingolipídeos/farmacologiaRESUMO
Type 2 diabetes (T2D) manifests from inadequate glucose control due to insulin resistance, hypoinsulinemia, and deteriorating pancreatic ß-cell function. The pro-inflammatory factor Activin has been implicated as a positive correlate of severity in T2D patients, and as a negative regulator of glucose uptake by skeletal muscle, and of pancreatic ß-cell phenotype in mice. Accordingly, we sought to determine whether intervention with the Activin antagonist Follistatin can ameliorate the diabetic pathology. Here, we report that an intravenous Follistatin gene delivery intervention with tropism for striated muscle reduced the serum concentrations of Activin B and improved glycemic control in the db/db mouse model of T2D. Treatment reversed the hyperglycemic progression with a corresponding reduction in the percentage of glycated-hemoglobin to levels similar to lean, healthy mice. Follistatin gene delivery promoted insulinemia and abundance of insulin-positive pancreatic ß-cells, even when treatment was administered to mice with advanced diabetes, supporting a mechanism for improved glycemic control associated with maintenance of functional ß-cells. Our data demonstrate that single-dose intravascular Follistatin gene delivery can ameliorate the diabetic progression and improve prognostic markers of disease. These findings are consistent with other observations of Activin-mediated mechanisms exerting deleterious effects in models of obesity and diabetes, and suggest that interventions that attenuate Activin signaling could help further understanding of T2D and the development of novel T2D therapeutics.
Assuntos
Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 2/terapia , Folistatina/genética , Técnicas de Transferência de Genes , Terapia Genética , Controle Glicêmico , Hiperglicemia/terapia , Administração Intravenosa , Animais , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Folistatina/administração & dosagem , Hiperglicemia/genética , Resistência à Insulina , CamundongosRESUMO
Adipose tissue is an energy store and a dynamic endocrine organ1,2. In particular, visceral adipose tissue (VAT) is critical for the regulation of systemic metabolism3,4. Impaired VAT function-for example, in obesity-is associated with insulin resistance and type 2 diabetes5,6. Regulatory T (Treg) cells that express the transcription factor FOXP3 are critical for limiting immune responses and suppressing tissue inflammation, including in the VAT7-9. Here we uncover pronounced sexual dimorphism in Treg cells in the VAT. Male VAT was enriched for Treg cells compared with female VAT, and Treg cells from male VAT were markedly different from their female counterparts in phenotype, transcriptional landscape and chromatin accessibility. Heightened inflammation in the male VAT facilitated the recruitment of Treg cells via the CCL2-CCR2 axis. Androgen regulated the differentiation of a unique IL-33-producing stromal cell population specific to the male VAT, which paralleled the local expansion of Treg cells. Sex hormones also regulated VAT inflammation, which shaped the transcriptional landscape of VAT-resident Treg cells in a BLIMP1 transcription factor-dependent manner. Overall, we find that sex-specific differences in Treg cells from VAT are determined by the tissue niche in a sex-hormone-dependent manner to limit adipose tissue inflammation.
Assuntos
Hormônios Esteroides Gonadais/metabolismo , Gordura Intra-Abdominal/imunologia , Caracteres Sexuais , Linfócitos T Reguladores/imunologia , Androgênios/metabolismo , Animais , Quimiocina CCL2/imunologia , Cromatina/genética , Feminino , Regulação da Expressão Gênica , Inflamação/imunologia , Inflamação/metabolismo , Interleucina-33/imunologia , Gordura Intra-Abdominal/metabolismo , Masculino , Camundongos , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , RNA-Seq , Receptores CCR2/metabolismo , Células Estromais/citologia , Células Estromais/imunologia , Células Estromais/metabolismo , Linfócitos T Reguladores/metabolismo , Transcrição GênicaRESUMO
Mitochondrial stress has been widely observed in diabetic kidney disease (DKD). Cyclophilin D (CypD) is a functional component of the mitochondrial permeability transition pore (mPTP) which allows the exchange of ions and solutes between the mitochondrial matrix to induce mitochondrial swelling and activation of cell death pathways. CypD has been successfully targeted in other disease contexts to improve mitochondrial function and reduced pathology. Two approaches were used to elucidate the role of CypD and the mPTP in DKD. Firstly, mice with a deletion of the gene encoding CypD (Ppif-/-) were rendered diabetic with streptozotocin (STZ) and followed for 24 weeks. Secondly, Alisporivir, a CypD inhibitor was administered to the db/db mouse model (5 mg/kg/day oral gavage for 16 weeks). Ppif-/- mice were not protected against diabetes-induced albuminuria and had greater glomerulosclerosis than their WT diabetic littermates. Renal hyperfiltration was lower in diabetic Ppif-/- as compared with WT mice. Similarly, Alisporivir did not improve renal function nor pathology in db/db mice as assessed by no change in albuminuria, KIM-1 excretion and glomerulosclerosis. Db/db mice exhibited changes in mitochondrial function, including elevated respiratory control ratio (RCR), reduced mitochondrial H2O2 generation and increased proximal tubular mitochondrial volume, but these were unaffected by Alisporivir treatment. Taken together, these studies indicate that CypD has a complex role in DKD and direct targeting of this component of the mPTP will likely not improve renal outcomes.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Nefropatias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Peptidil-Prolil Isomerase F/metabolismo , Albuminúria/genética , Albuminúria/metabolismo , Animais , Peptidil-Prolil Isomerase F/antagonistas & inibidores , Peptidil-Prolil Isomerase F/genética , Ciclosporina/farmacologia , Diabetes Mellitus Experimental/genética , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Peróxido de Hidrogênio/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Nefropatias/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Poro de Transição de Permeabilidade Mitocondrial , ATPases Translocadoras de Prótons/metabolismoRESUMO
The sequelae of diabetes include microvascular complications such as diabetic kidney disease (DKD), which involves glucose-mediated renal injury associated with a disruption in mitochondrial metabolic agility, inflammation, and fibrosis. We explored the role of the innate immune complement component C5a, a potent mediator of inflammation, in the pathogenesis of DKD in clinical and experimental diabetes. Marked systemic elevation in C5a activity was demonstrated in patients with diabetes; conventional renoprotective agents did not therapeutically target this elevation. C5a and its receptor (C5aR1) were upregulated early in the disease process and prior to manifest kidney injury in several diverse rodent models of diabetes. Genetic deletion of C5aR1 in mice conferred protection against diabetes-induced renal injury. Transcriptomic profiling of kidney revealed diabetes-induced downregulation of pathways involved in mitochondrial fatty acid metabolism. Interrogation of the lipidomics signature revealed abnormal cardiolipin remodeling in diabetic kidneys, a cardinal sign of disrupted mitochondrial architecture and bioenergetics. In vivo delivery of an orally active inhibitor of C5aR1 (PMX53) reversed the phenotypic changes and normalized the renal mitochondrial fatty acid profile, cardiolipin remodeling, and citric acid cycle intermediates. In vitro exposure of human renal proximal tubular epithelial cells to C5a led to altered mitochondrial respiratory function and reactive oxygen species generation. These experiments provide evidence for a pivotal role of the C5a/C5aR1 axis in propagating renal injury in the development of DKD by disrupting mitochondrial agility, thereby establishing a new immunometabolic signaling pathway in DKD.
Assuntos
Complemento C5a/fisiologia , Diabetes Mellitus Experimental , Nefropatias Diabéticas , Rim/patologia , Mitocôndrias/metabolismo , Animais , Células Cultivadas , Complemento C5a/genética , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Metabolismo Energético/genética , Fibrose/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ratos , Ratos Sprague-Dawley , Receptor da Anafilatoxina C5a/fisiologia , Transdução de SinaisRESUMO
The gp130 receptor cytokines IL-6 and CNTF improve metabolic homeostasis but have limited therapeutic use for the treatment of type 2 diabetes. Accordingly, we engineered the gp130 ligand IC7Fc, in which one gp130-binding site is removed from IL-6 and replaced with the LIF-receptor-binding site from CNTF, fused with the Fc domain of immunoglobulin G, creating a cytokine with CNTF-like, but IL-6-receptor-dependent, signalling. Here we show that IC7Fc improves glucose tolerance and hyperglycaemia and prevents weight gain and liver steatosis in mice. In addition, IC7Fc either increases, or prevents the loss of, skeletal muscle mass by activation of the transcriptional regulator YAP1. In human-cell-based assays, and in non-human primates, IC7Fc treatment results in no signs of inflammation or immunogenicity. Thus, IC7Fc is a realistic next-generation biological agent for the treatment of type 2 diabetes and muscle atrophy, disorders that are currently pandemic.