RESUMO
OBJECTIVE: To compare the clinical and radiological outcomes of patients who underwent rotator cuff repair (RCR) concomitant with long head of the biceps tendon (LHBT) tenotomy or subpectoral mini-open tenodesis. METHODS: Prospectively collected data was reviewed on 154 patients, who underwent a LHBT procedure (tenotomy or tenodesis) concomitant with RCR between January 2010 and January 2017. The exclusion criteria were irreparable massive rotator cuff tear, rotator cuff partial tear, subscapular tendon tear, glenohumeral arthritis, and prior shoulder surgery. The two patient groups are as follows: RCR + Tenotomy (Group A) and RCR + Subpectoral mini-open tenodesis (Group B). The visual analog scale (VAS) for pain, Constant Score scale, American Shoulder and Elbow Surgeons (ASES) scores, and the Disabilities of the Arm, Shoulder and Hand (DASH) scores preoperatively and 1 month, 3 months, 6 months, 1 year postoperatively and the latest out-patient clinic were compared between the two groups. RESULTS: Ninety-two patients in Group A and 62 patients in Group B completed the follow-up, with a median follow-up time of 27 and 42 months respectively. At the final follow-up, the VAS, Constant, ASES, and DASH scores in Group A were 0.1 ± 0.2, 87.0 ± 12.8, 96.4 ± 4.3 and 6.6 ± 4.8 respectively, and the VAS, Constant, ASES, and DASH scores in Group B were 0.1 ± 0.3, 92.5 ± 3.9, 96.3 ± 3.6 and 2.9 ± 1.3 respectively. Clinical evaluation scales showed satisfactory results in both groups, and there were no statistically significant differences between the two groups at the same follow-up time. Popeye sign was detected in one case of Group A (1.1%) and in one case of Group B (1.6%, P > 0.05). CONCLUSION: Both tenotomy and subpectoral mini-open tenodesis are effective for concomitant lesions of the LHBT in patients with reparable rotator cuff tears, and subpectoral mini-open tenodesis of the LHBT does not provide any significant clinical or functional improvement than isolated tenotomy.
Assuntos
Músculo Esquelético/cirurgia , Procedimentos de Cirurgia Plástica/métodos , Lesões do Manguito Rotador/cirurgia , Traumatismos dos Tendões/cirurgia , Tenodese/métodos , Tenotomia/métodos , Idoso , Artroscopia , Avaliação da Deficiência , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/lesões , Medição da Dor , Estudos ProspectivosRESUMO
PURPOSE: To compare the clinical outcomes and complications and radiographic outcomes of the two different surgical strategies (arthrodesis in situ and arthrodesis following reduction) for the surgical management of spondylolisthesis. METHODS: After systematic search the PubMed, Ovid MEDLINE, Cochrane, and Embase databases, comparative studies were selected according to eligibility criteria. Checklists by Furlan and by The Newcastle-Ottawa quality assessment scale (NOS scale) were used to evaluate the risk of bias of the included randomized clinical trials (RCTs) and nonrandomized controlled studies, respectively. The final strength of evidence was expressed as different levels recommended by the GRADE Working Group. RESULTS: Three RCTs. and nine comparative observational studies were identified. Low-quality evidence indicated that reduction group (RG) was not more effective than fusion in situ group for clinical satisfaction (OR 0.77, 95% CI 0.39-1.54, P = 0.46). and neurologic complication rate (OR 0.89, 95 CI 0.38-2.03, P = 0.78). In secondary outcomes, Low-quality evidence indicated that RG improved fusion rate (OR 2.66, 95% CI 1.15-6.14, P = 0.02). There was no significant difference in the other complication rate (OR 0.89, 95% CI 0.44-1.79, P = 0.63) and blood loss (WMD 14.22, 95% CI -9.53-37.79, P = 0.24) between two groups. Statistical difference was found between the two groups with regard to slipping angle (WMD -6.33, 95% CI -12.60 to -0.06, P = 0.05). CONCLUSIONS: There was no definite benefit of reduction over fusion in situ in clinical satisfaction rate and neurologic complication rate. The fusion rate significantly improved while the slipping angle considerably decreased postoperation in reduction group.
Assuntos
Fusão Vertebral/métodos , Espondilolistese/cirurgia , Feminino , HumanosRESUMO
Phenotypic divergence in diet-induced obesity (DIO) and hepatic steatosis has been reported in two independently generated lines of L-Fabp(-/-) mice [New Jersey (NJ) L-Fabp(-/-) vs. Washington University (WU) L-Fabp(-/-) mice]. We performed side-by-side studies to examine differences between the lines and investigate the role of genetic background, intestinal microbiota, sex, and diet in the divergent phenotypes. Fasting-induced steatosis was attenuated in both L-Fabp(-/-) lines compared with C57BL/6J controls, with restoration of hepatic triglyceride levels following adenoviral L-Fabp rescue. Both lines were protected against DIO after high-saturated-fat diet feeding. Hepatic steatosis was attenuated in WU but not NJ L-Fabp(-/-) mice, although this difference between the lines disappeared upon antibiotic treatment and cohousing. In contrast, there was phenotypic divergence in L-Fabp(-/-) mice fed a high cocoa butter fat diet, with WU L-Fabp(-/-) mice, but not NJ L-Fabp(-/-) mice, showing protection against both DIO and hepatic steatosis, with some sex-dependent (female > male) differences. Dense mapping revealed no evidence of unintended targeting, duplications, or deletions surrounding the Fabp1 locus in either line and only minor differences in mRNA expression of genes located near the targeted allele. However, a C57BL/6 substrain screen showed that the NJ L-Fabp(-/-) line contains â¼40% C57BL/6N genomic DNA, despite reports that these mice were backcrossed six generations. Overall, these findings suggest that some of the phenotypic divergence between the two L-Fabp(-/-) lines may reflect unanticipated differences in genetic background, underscoring the importance of genetic background in phenotypic characterization.
Assuntos
Proteínas de Ligação a Ácido Graxo/metabolismo , Regulação da Expressão Gênica/fisiologia , Animais , Antibacterianos/administração & dosagem , Antibacterianos/farmacologia , Gorduras na Dieta , Proteínas de Ligação a Ácido Graxo/genética , Fígado Gorduroso/etiologia , Fígado Gorduroso/genética , Fígado Gorduroso/prevenção & controle , Feminino , Privação de Alimentos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/genética , Obesidade/metabolismo , Obesidade/prevenção & controle , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
HuR is a ubiquitous nucleocytoplasmic RNA-binding protein that exerts pleiotropic effects on cell growth and tumorigenesis. In this study, we explored the impact of conditional, tissue-specific genetic deletion of HuR on intestinal growth and tumorigenesis in mice. Mice lacking intestinal expression of HuR (Hur (IKO) mice) displayed reduced levels of cell proliferation in the small intestine and increased sensitivity to doxorubicin-induced acute intestinal injury, as evidenced by decreased villus height and a compensatory shift in proliferating cells. In the context of Apc(min/+) mice, a transgenic model of intestinal tumorigenesis, intestinal deletion of the HuR gene caused a three-fold decrease in tumor burden characterized by reduced proliferation, increased apoptosis, and decreased expression of transcripts encoding antiapoptotic HuR target RNAs. Similarly, Hur(IKO) mice subjected to an inflammatory colon carcinogenesis protocol [azoxymethane and dextran sodium sulfate (AOM-DSS) administration] exhibited a two-fold decrease in tumor burden. Hur(IKO) mice showed no change in ileal Asbt expression, fecal bile acid excretion, or enterohepatic pool size that might explain the phenotype. Moreover, none of the HuR targets identified in Apc(min/+)Hur(IKO) were altered in AOM-DSS-treated Hur(IKO) mice, the latter of which exhibited increased apoptosis of colonic epithelial cells, where elevation of a unique set of HuR-targeted proapoptotic factors was documented. Taken together, our results promote the concept of epithelial HuR as a contextual modifier of proapoptotic gene expression in intestinal cancers, acting independently of bile acid metabolism to promote cancer. In the small intestine, epithelial HuR promotes expression of prosurvival transcripts that support Wnt-dependent tumorigenesis, whereas in the large intestine epithelial HuR indirectly downregulates certain proapoptotic RNAs to attenuate colitis-associated cancer. Cancer Res; 74(18); 5322-35. ©2014 AACR.
Assuntos
Neoplasias do Colo/patologia , Proteínas ELAV/fisiologia , Mucosa Intestinal/patologia , Neoplasias Intestinais/patologia , Animais , Apoptose/fisiologia , Processos de Crescimento Celular/fisiologia , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Modelos Animais de Doenças , Proteínas ELAV/genética , Proteínas ELAV/metabolismo , Mucosa Intestinal/metabolismo , Neoplasias Intestinais/genética , Neoplasias Intestinais/metabolismo , Camundongos , Camundongos KnockoutRESUMO
Evidence suggests a relationship between dietary fat intake, obesity, and colorectal cancer, implying a role for fatty acid metabolism in intestinal tumorigenesis that is incompletely understood. Liver fatty acid-binding protein (L-Fabp), a dominant intestinal fatty acid-binding protein, regulates intestinal fatty acid trafficking and metabolism, and L-Fabp deletion attenuates diet-induced obesity. Here, we examined whether changes in intestinal fatty acid metabolism following L-Fabp deletion modify adenoma development in Apc(Min)(/+) mice. Compound L-Fabp(-/-)Apc(Min)(/+) mice were generated and fed a 10% fat diet balanced equally between saturated, monounsaturated, and polyunsaturated fat. L-Fabp(-/-)Apc(Min)(/+) mice displayed significant reductions in adenoma number and total polyp area compared with Apc(Min)(/+)controls, reflecting a significant shift in distribution toward smaller polyps. Adenomas from L-Fabp(-/-)Apc(Min)(/+) mice exhibited reductions in cellular proliferation, high-grade dysplasia, and nuclear ß-catenin translocation. Intestinal fatty acid content was increased in L-Fabp(-/-)Apc(Min)(/+) mice, and lipidomic profiling of intestinal mucosa revealed significant shifts to polyunsaturated fatty acid species with reduced saturated fatty acid species. L-Fabp(-/-)Apc(Min)(/+) mice also showed corresponding changes in mRNA expression of enzymes involved in fatty acid elongation and desaturation. Furthermore, adenomas from L-Fabp(-/-)Apc(Min)(/+) mice displayed significant reductions in mRNA abundance of nuclear hormone receptors involved in cellular proliferation and in enzymes involved in lipogenesis. These findings collectively implicate L-Fabp as an important genetic modifier of intestinal tumorigenesis, and identify fatty acid trafficking and metabolic compartmentalization as an important pathway linking dietary fat intake, obesity, and intestinal tumor formation.
Assuntos
Adenoma/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Ácidos Graxos/metabolismo , Regulação Neoplásica da Expressão Gênica , Mucosa Intestinal/metabolismo , Animais , Proliferação de Células , Gorduras na Dieta , Dinoprostona/metabolismo , Feminino , Deleção de Genes , Genótipo , Imuno-Histoquímica , Lipídeos/química , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
BACKGROUND: Gut derived lipid factors have been implicated in systemic injury and inflammation but the precise pathways involved are unknown. In addition, dietary fat intake and obesity are independent risk factors for the development of colorectal cancer. Here we studied the severity of experimental colitis and the development of colitis associated cancer (CAC) in mice with an inducible block in chylomicron secretion and fat malabsorption, following intestine-specific deletion of microsomal triglyceride transfer protein (Mttp-IKO). METHODOLOGY/PRINCIPAL FINDINGS: Mttp-IKO mice exhibited more severe injury with â¼90% mortality following dextran sodium sulfate (DSS) induced colitis, compared to <20% in controls. Intestinal permeability was increased in Mttp-IKO mice compared to controls, both at baseline and after DSS administration, in association with increased circulating levels of TNFα. DSS treatment increased colonic mRNA expression of IL-1ß and IL-17A as well as inflammasome expression in both genotypes, but the abundance of TNFα was selectively increased in DSS treated Mttp-IKO mice. There was a 2-fold increase in colonic tumor burden in Mttp-IKO mice following azoxymethane/DSS treatment, which was associated with increased colonic inflammation as well as alterations in cytokine expression. To examine the pathways by which alterations in fatty acid abundance might interact with cytokine signaling to regulate colonic epithelial growth, we used primary murine myofibroblasts to demonstrate that palmitate induced expression of amphiregulin and epiregulin and augmented the increase in both of these growth mediators when added to IL-1ßor to TNFα. CONCLUSIONS: These studies demonstrate that Mttp-IKO mice, despite absorbing virtually no dietary fat, exhibit augmented fatty acid dependent signaling that in turn exacerbates colonic injury and increases tumor formation.
Assuntos
Proteínas de Transporte/genética , Colite/patologia , Neoplasias do Colo/patologia , Intestinos/patologia , Animais , Azoximetano/toxicidade , Proteínas de Transporte/metabolismo , Células Cultivadas , Colite/induzido quimicamente , Colite/complicações , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/genética , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Fezes/química , Interleucina-17/genética , Interleucina-17/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miofibroblastos/citologia , Miofibroblastos/metabolismo , Índice de Gravidade de Doença , Carga Tumoral/genética , Fator de Necrose Tumoral alfa/sangueRESUMO
Intestinal apolipoprotein B (apoB) mRNA undergoes C-to-U editing, mediated by the catalytic deaminase apobec-1, which results in translation of apoB48. Apobec1(-/-) mice produce only apoB100 and secrete larger chylomicron particles than those observed in wild-type (WT) mice. Here we show that transgenic rescue of intestinal apobec-1 expression (Apobec1(Int/O)) restores C-to-U RNA editing of apoB mRNA in vivo, including the canonical site at position 6666 and also at approximately 20 other newly identified downstream sites present in WT mice. The small intestine of Apobec1(Int/O) mice produces only apoB48, and the liver produces only apoB100. Serum chylomicron particles were smaller in Apobec1(Int/O) mice compared with those from Apobec1(-/-) mice, and the predominant fraction of serum apoB48 in Apobec1(Int/O) mice migrated in lipoproteins smaller than chylomicrons, even when these mice were fed a high-fat diet. Because apoB48 arises exclusively from the intestine in Apobec1(Int/O) mice and intestinal apoB48 synthesis and secretion rates were comparable to WT mice, we were able to infer the major sites of origin of serum apoB48 in WT mice. Our findings imply that less than 25% of serum apoB48 in WT mice arises from the intestine, with the majority originating from the liver.
Assuntos
Apolipoproteínas B/genética , Quilomícrons/biossíntese , Citidina Desaminase/deficiência , Intestino Delgado/metabolismo , Edição de RNA , Desaminase APOBEC-1 , Animais , Apolipoproteínas B/metabolismo , Cromatografia Líquida de Alta Pressão , Quilomícrons/sangue , Quilomícrons/química , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica , Especificidade de Órgãos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Apobec-1 complementation factor (ACF) is the RNA binding subunit of a core complex that mediates C to U RNA editing of apolipoprotein B (apoB) mRNA. Targeted deletion of the murine Acf gene is early embryonic lethal and Acf(-/-) blastocysts fail to implant and proliferate, suggesting that ACF plays a key role in cell growth and differentiation. Here we demonstrate that heterozygous Acf(+/-) mice exhibit decreased proliferation and impaired liver mass restitution following partial hepatectomy (PH). To pursue the mechanism of impaired liver regeneration we examined activation of interleukin-6 (IL-6) a key cytokine required for induction of hepatocyte proliferation following PH. Peak induction of hepatic IL-6 mRNA abundance post PH was attenuated >80% in heterozygous Acf(+/-) mice, along with decreased serum IL-6 levels. IL-6 secretion from isolated Kupffer cells (KC) was 2-fold greater in wild-type compared with heterozygous Acf(+/-) mice. Recombinant ACF bound an AU-rich region in the IL-6 3'-untranslated region with high affinity and IL-6 mRNA half-life was significantly shorter in KC isolated from Acf(+/-) mice compared with wild-type controls. These findings suggest that ACF regulates liver regeneration following PH at least in part by controlling the stability of IL-6 mRNA. The results further suggest a new RNA target and an unanticipated physiological function for ACF beyond apoB RNA editing.
Assuntos
Citidina Desaminase/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Interleucina-6/metabolismo , Regeneração Hepática , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas , Desaminase APOBEC-1 , Animais , Proliferação de Células , Fibroblastos/metabolismo , Deleção de Genes , Hepatócitos/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/fisiologia , Heterozigoto , Fígado/metabolismo , CamundongosRESUMO
Quantitative trait mapping in mice identified a susceptibility locus for gallstones (Lith6) spanning the Apobec-1 locus, the structural gene encoding the RNA-specific cytidine deaminase responsible for production of apolipoprotein B48 in mammalian small intestine and rodent liver. This observation prompted us to compare dietary gallstone susceptibility in Apobec-1(-/-) mice and congenic C57BL/6 wild type controls. When fed a lithogenic diet (LD) for 2 weeks, 90% Apobec-1(-/-) mice developed solid gallstones in comparison with 16% wild type controls. LD-fed Apobec-1(-/-) mice demonstrated increased biliary cholesterol secretion as well as increased cholesterol saturation and bile acid hydrophobicity indices. These changes occurred despite a relative decrease in cholesterol absorption in LD-fed Apobec-1(-/-) mice. Among the possible mechanisms to account for this phenotype, expression of Cyp7a1 mRNA and protein were significantly decreased in chow-fed Apobec-1(-/-) mice, decreasing further in LD-fed animals. Cyp7a1 transcription in hepatocyte nuclei, however, was unchanged in Apobec-1(-/-) mice, excluding transcriptional repression as a potential mechanism for decreased Cyp7a1 expression. We demonstrated that APOBEC-1 binds to AU-rich regions of the 3'-untranslated region of the Cyp7a1 transcript, containing the UUUN(A/U)U consensus motif, using both UV cross-linking to recombinant APOBEC-1 and in vivo RNA co-immunoprecipitation. In vivo Apobec-1-dependent modulation of Cyp7a1 expression was further confirmed following adenovirus-Apobec-1 administration to chow-fed Apobec-1(-/-) mice, which rescued Cyp7a1 gene expression. Taken together, the findings suggest that the AU-rich RNA binding-protein Apobec-1 mediates post-transcriptional regulation of murine Cyp7a1 expression and influences susceptibility to diet-induced gallstone formation.
Assuntos
Ácidos e Sais Biliares/metabolismo , Colesterol 7-alfa-Hidroxilase/genética , Colesterol 7-alfa-Hidroxilase/metabolismo , Citidina Desaminase/deficiência , Cálculos Biliares/etiologia , Regiões 3' não Traduzidas , Desaminase APOBEC-1 , Animais , Animais Geneticamente Modificados , Sequência de Bases , Sítios de Ligação/genética , Colesterol/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Dieta/efeitos adversos , Cálculos Biliares/genética , Cálculos Biliares/metabolismo , Cálculos Biliares/patologia , Hepatócitos/metabolismo , Masculino , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Microsomal triglyceride transfer protein (Mttp) is a key player in the assembly and secretion of hepatic very low density lipoproteins (VLDL). Here we determined the effects of Mttp overexpression on hepatic triglyceride (TG) and VLDL secretion in leptin-deficient (ob/ob) mice, specifically in relation to apolipoproteinB (apoB) isoforms. We crossed Apobec1(-/-) mice with congenic ob/ob mice to generate apoB100-only ob/ob mice (A-ob/ob). The obesity phenotype in both genotypes was similar, but A-ob/ob mice had greater hepatic TG content. Administration of recombinant adenovirus expressing murine Mttp cDNA (Ad-mMTP) increased hepatic Mttp content and activity and increased hepatic VLDL-TG secretion in A-ob/ob mice. However, despite equivalent overexpression of Mttp, there was no change in VLDL-TG secretion in ob/ob mice in a wild-type Apobec1 background. Metabolic labeling studies in primary hepatocytes from A-ob/ob mice demonstrated that Ad-mMTP increased triglyceride secretion without changing the synthesis and secretion of apoB100, suggesting greater incorporation of TG into existing VLDL particles rather than increased particle number. Ad-mMTP administration failed to increase hepatic VLDL secretion in lean Apobec1(-/-) mice or controls. By contrast, VLDL secretion increased and hepatic TG content decreased following Ad-mMTP administration to human APOB transgenic mice crossed into the Apobec1(-/-) line. These findings demonstrate that Ad-mMTP increases murine hepatic VLDL-TG secretion only in the apoB100 background, and even then only in situations with either increased hepatic TG accumulation or increased apoB100 expression.
Assuntos
Apolipoproteína B-100/fisiologia , Proteínas de Transporte/fisiologia , Lipoproteínas VLDL/metabolismo , Triglicerídeos/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Obesos , Microssomos Hepáticos/metabolismoRESUMO
Liver fatty acid (FA)-binding protein (L-Fabp), a cytoplasmic protein expressed in liver and small intestine, regulates FA trafficking in vitro and plays an important role in diet-induced obesity. We observed that L-Fabp(-/-) mice are protected against Western diet-induced obesity and hepatic steatosis. These findings are in conflict, however, with another report of exaggerated obesity and increased hepatic steatosis in female L-Fabp(-/-) mice fed a cholesterol-supplemented diet. To resolve this apparent paradox, we fed female L-Fabp(-/-) mice two different cholesterol-supplemented low-fat diets and discovered (on both diets) lower body weight in L-Fabp(-/-) mice than in congenic wild-type C57BL/6J controls and similar or reduced hepatic triglyceride content. We extended these comparisons to mice fed low-cholesterol, high-fat diets. Female L-Fabp(-/-) mice fed a high-saturated fat (SF) diet were dramatically protected against obesity and hepatic steatosis, whereas weight gain and hepatic lipid content were indistinguishable between mice fed a high-polyunsaturated FA (PUFA) diet and control mice. These findings demonstrate that L-Fabp functions as a metabolic sensor with a distinct hierarchy of FA sensitivity. We further conclude that cholesterol supplementation does not induce an obesity phenotype in L-Fabp(-/-) mice, nor does it play a significant role in the protection against Western diet-induced obesity in this background.
Assuntos
Colesterol na Dieta/administração & dosagem , Proteínas de Ligação a Ácido Graxo/metabolismo , Ácidos Graxos Insaturados/administração & dosagem , Ácidos Graxos/administração & dosagem , Fígado Gorduroso/prevenção & controle , Metabolismo dos Lipídeos , Fígado/metabolismo , Obesidade/prevenção & controle , Animais , Peso Corporal , Colesterol na Dieta/metabolismo , Modelos Animais de Doenças , Proteínas de Ligação a Ácido Graxo/deficiência , Proteínas de Ligação a Ácido Graxo/genética , Ácidos Graxos/metabolismo , Ácidos Graxos Insaturados/metabolismo , Fígado Gorduroso/etiologia , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Feminino , Metabolismo dos Lipídeos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Obesidade/induzido quimicamente , Obesidade/complicações , Obesidade/genética , Obesidade/metabolismo , RNA Mensageiro/metabolismo , Fatores de Tempo , Triglicerídeos/metabolismoRESUMO
The RNA-specific cytidine deaminase apobec-1 is an AU-rich RNA binding protein that binds the 3' untranslated region (UTR) of cyclooxygenase-2 (Cox-2) mRNA and stabilizes its turnover in vitro. Cox-2 overexpression accompanies intestinal adenoma formation in both humans and mice. Evidence from both genetic deletion studies as well as from pharmacologic inhibition has implicated Cox-2 in the development of intestinal adenomas in experimental animals and in adenomas and colorectal cancer in humans. Here, we show that small intestinal adenoma formation is dramatically reduced in compound Apc(min/+) apobec-1(-/-) mice when compared with the parental Apc(min/+) strain. This reduced tumor burden was found in association with increased small intestinal apoptosis and reduced proliferation in small intestinal crypt-villus units from compound Apc(min/+) apobec-1(-/-) mice. Intestinal adenomas from compound Apc(min/+) apobec-1(-/-) mice showed a <2-fold increase in Cox-2 mRNA abundance and reduced prostaglandin E(2) content compared with adenomas from the parental Apc(min/+) strain. In addition, there was reduced expression in adenomas from compound Apc(min/+) apobec-1(-/-) mice of other mRNAs (including epidermal growth factor receptor, peroxisome proliferator-activated receptor delta, prostaglandin receptor EP4, and c-myc), each containing the apobec-1 consensus binding site within their 3'-UTR. Adenovirus-mediated apobec-1 introduction into HCA-7 (colorectal cancer) cells showed a dose-dependent increase in Cox-2 protein and stabilization of endogenous Cox-2 mRNA. These findings suggest that deletion of apobec-1, by modulating expression of AU-rich RNA targets, provides an important mechanism for attenuating a dominant genetic restriction point in intestinal adenoma formation.
Assuntos
Adenoma/enzimologia , Citidina Desaminase/deficiência , Neoplasias Intestinais/enzimologia , Desaminase APOBEC-1 , Adenoma/genética , Animais , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/genética , Citidina Desaminase/biossíntese , Citidina Desaminase/genética , Genes APC , Humanos , Neoplasias Intestinais/genética , Intestino Delgado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
Mammalian small intestinal lipid absorption requires the coordinated interactions of apolipoprotein B (apoB) and the microsomal triglyceride transfer protein (Mttp). The observation that apoB100 displays greater dependence on Mttp availability than does apoB48 prompted us to examine the phenotype of Mttp deletion in an Apobec-1(-/-) background (i.e. apoB100 Mttp-IKO). 20% apoB100 Mttp-IKO mice died on a chow diet, and >90% died following high fat feeding (versus 0 and 11% apoB48 Mttp-IKO mice, respectively). Intestinal adaptation occurred in apoB48 Mttp-IKO mice in response to high fat feeding, evidenced by increased bromodeoxyuridine incorporation and villus lengthening, changes that did not occur in apoB100 Mttp-IKO mice. There was an exaggerated unfolded protein response (UPR), which became more pronounced in apoB100 Mttp-IKO mice. To examine the role of endoplasmic reticulum stress and the UPR in the lipotoxic effects of Mttp deletion, we administered tauroursodeoxycholate to apoB100 Mttp-IKO mice upon initiation of high fat feeding. Tauroursodeoxycholate administration abrogated the UPR but produced an unexpected acceleration in the onset of lethality in apoB100 Mttp-IKO mice. The findings demonstrate that there is activation of the UPR with lethal lipotoxicity in conditional intestinal apoB100 Mttp-IKO mice. Together the data provide the first plausible biological evidence for a survival advantage for mammalian intestinal apoB mRNA editing.
Assuntos
Apolipoproteína B-100/metabolismo , Apolipoproteína B-48/metabolismo , Proteínas de Transporte/metabolismo , Citidina Desaminase/deficiência , Citidina Desaminase/metabolismo , Mucosa Intestinal/metabolismo , Desaminase APOBEC-1 , Ração Animal , Animais , Apolipoproteína B-100/genética , Apolipoproteína B-48/genética , Proteínas de Transporte/genética , Citidina Desaminase/genética , Retículo Endoplasmático/metabolismo , Gorduras/farmacologia , Genótipo , Intestinos/efeitos dos fármacos , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , Taxa de SobrevidaRESUMO
apobec-1 complementation factor (ACF) is an hnRNP family member which functions as the obligate RNA binding subunit of the core enzyme mediating C-to-U editing of the nuclear apolipoprotein B (apoB) transcript. ACF binds to both apoB RNA and apobec-1, the catalytic cytidine deaminase, which then results in site-specific posttranscriptional editing of apoB mRNA. Targeted deletion of apobec1 eliminates C-to-U editing of apoB mRNA but is otherwise well tolerated. However, the functions and potential targets of ACF beyond apoB mRNA editing are unknown. Here we report the results of generating acf knockout mice using homologous recombination. While heterozygous acf(+/)(-) mice were apparently healthy and fertile, no viable acf(-)(/)(-) mice were identified. Mutant acf(-)(/)(-) embryos were detectable only until the blastocyst (embryonic day 3.5 [E3.5]) stage. No acf(-)(/)(-) blastocysts were detectable following implantation at E4.5, and isolated acf(-)(/)(-) blastocysts failed to proliferate in vitro. Small interfering RNA knockdown of ACF in either rat (apobec-1-expressing) or human (apobec-1-deficient) hepatoma cells decreased ACF protein expression and induced a commensurate increase in apoptosis. Taken together, these data suggest that ACF plays a crucial role, which is independent of apobec-1 expression, in cell survival, particularly during early embryonic development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Teste de Complementação Genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Proteínas de Ligação a RNA/genética , Animais , Apolipoproteínas B/metabolismo , Apoptose , Cruzamentos Genéticos , Citidina Desaminase/metabolismo , Implantação do Embrião , Éxons , Deleção de Genes , Vetores Genéticos , Genótipo , Ribonucleoproteínas Nucleares Heterogêneas/fisiologia , Heterozigoto , Homozigoto , Humanos , Íntrons , Rim/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Genéticos , Mutação , Fenótipo , Ligação Proteica , RNA/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Recombinação Genética , Transcrição GênicaRESUMO
Recent studies have examined the role of the LDL receptor (LDLR) in regulating murine hepatic lipoprotein production and apolipoprotein B (apoB) secretion, with divergent conclusions from in vivo versus in vitro approaches. We have re-examined this question, both in vivo and in vitro, using apobec-1-/- mice to model the pattern of human hepatic apoB-100 secretion. Hepatic triglyceride production in vivo (using Triton WR-1339) was unchanged in wild-type (WT) C57BL/6, apobec-1-/-, ldlr-/-, and [apobec-1-/-, ldlr-/-] mice, while apoB-100 production (using [35S]methionine incorporation) was increased > 2-fold in [apobec-1-/-, ldlr-/-] mice. Although > 90% of newly synthesized apoB floated within the d < 1.006 fraction of serum from all genotypes, fast-performance liquid chromatography separation revealed that nascent triglyceride-rich particles from [apobec-1-/-, ldlr-/-] mice, but not WT, apobec-1-/-, or ldlr-/- mice, distributed into smaller (intermediate and LDL-sized) particles. Studies in isolated hepatocytes from these different genotypes confirmed secretion of smaller particles exclusively from [apobec-1-/-, ldlr-/-] mice, and pulse-chase analysis demonstrated increased secretion of apoB-100 with virtual elimination of posttranslational degradation. These results directly support the suggestion that the LDLR regulates hepatic apoB-100 production and modulates secretion of small, triglyceride-rich particles, both in vivo and in vitro.
Assuntos
Citidina Desaminase/genética , Lipoproteínas/metabolismo , Fígado/metabolismo , Receptores de LDL/metabolismo , Desaminase APOBEC-1 , Animais , Apolipoproteínas B/metabolismo , Genótipo , Hepatócitos/metabolismo , Metabolismo dos Lipídeos , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tamanho da Partícula , Receptores de LDL/genética , Triglicerídeos/metabolismoRESUMO
Liver fatty acid-binding protein (L-Fabp) is an abundant cytosolic lipid-binding protein with broad substrate specificity, expressed in mammalian enterocytes and hepatocytes. We have generated mice with a targeted deletion of the endogenous L-Fabp gene and have characterized their response to alterations in hepatic fatty acid flux following prolonged fasting. Chow-fed L-Fabp-/- mice were indistinguishable from wild-type littermates with regard to growth, serum and tissue lipid profiles, and fatty acid distribution within hepatic complex lipid species. In response to 48-h fasting, however, wild-type mice demonstrated a approximately 10-fold increase in hepatic triglyceride content while L-Fabp-/- mice demonstrated only a 2-fold increase. Hepatic VLDL secretion was decreased in L-Fabp-/- mice suggesting that the decreased accumulation of hepatic triglyceride was not the result of increased secretion. Fatty acid oxidation, as inferred from serum beta-hydroxybutyrate levels, was increased in response to fasting, although the increase in L-Fabp-/- mice was significantly reduced in comparison to wild-type controls, despite comparable induction of PPAR alpha target genes. Studies in primary hepatocytes revealed indistinguishable initial rates of oleate uptake, but longer intervals revealed reduced rates of uptake in fasted L-Fabp-/- mice. Oleate incorporation into cellular triglyceride and diacylglycerol was reduced in L-Fabp-/- mice although incorporation into phospholipid and cholesterol ester was no different than wild-type controls. These data point to an inducible defect in fatty acid utilization in fasted L-Fabp-/- mice that involves targeting of substrate for use in triglyceride metabolism.
Assuntos
Proteínas de Transporte/fisiologia , Ácidos Graxos/metabolismo , Deleção de Genes , Fígado/metabolismo , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Triglicerídeos/metabolismo , Adaptação Fisiológica , Tecido Adiposo/metabolismo , Animais , Proteínas de Transporte/genética , Jejum , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Lipoproteínas VLDL/metabolismo , Fígado/citologia , Camundongos , Camundongos Knockout , Ácido Oleico/metabolismo , OxirreduçãoRESUMO
Mammalian enterocytes express apolipoprotein (apo)B-48, which is produced after posttranscriptional RNA editing of the nuclear apoB-100 transcript by the catalytic deaminase apobec-1. Earlier studies in apobec-1-/- mice revealed an apoB-100-only lipoprotein profile but no gross defects in triglyceride absorption. However, subtle defects may have been obscured by the mixed genetic background. In addition, the intrinsic susceptibility to proteolytic degradation of intestinal apoB-100 and apoB-48 has been questioned. Accordingly, we examined triglyceride absorption, intestinal apoB expression, and lipoprotein secretion in apobec-1-/- mice backcrossed into a C57BL/6 background. Inbred apobec-1-/- mice absorb triglyceride normally, yet secrete triglyceride-rich lipoproteins more slowly than wild-type congenic controls. There was comparable induction of apoB synthesis in response to fat feeding in both genotypes, but apoB-100 was preferentially retained and more extensively degraded than apoB-48. By contrast, synthesis, secretion, and content of apo A-IV were indistinguishable in apobec-1-/- and wild-type mice with 100% recovery, suggesting no degradation of this apoprotein in either genotype. Newly secreted lipoproteins from isolated enterocytes of wild-type mice revealed apoB-48 in both high-density lipoproteins and very low-density lipoproteins. By contrast, apobec-1-/- mice secreted apoB-100-containing particles that were almost exclusively in the low and very low-density lipoproteins range with no apoB-100-containing high-density lipoproteins. These studies establish the existence of preferential degradation of intestinal apoB-100 and subtle defects in triglyceride secretion in apobec-1-/- mice, coupled with a shift to the production of larger particles, findings that suggest an important divergence in intestinal lipoprotein assembly pathways with the different isoforms of apoB.