RESUMO
Tesaglitazar was developed as a dual peroxisome proliferator-activated receptor (PPARα/γ). To support the clinical program, a hamster carcinogenicity study was performed. The only neoplastic findings possibly related to treatment with tesaglitazar were low incidences of hemangioma and hemangiosarcoma in the liver of male animals. A high-power, two-year investigative study with interim necropsies was performed to further elucidate these findings. Treatment with tesaglitazar resulted in changes typical for exaggerated PPARα pharmacology in rodents, such as hepatocellular hypertrophy and hepatocellular carcinoma, but not an increased frequency of hemangiosarcomas. At the highest dose level, there was an increased incidence of sinusoidal dilatation and hemangiomas. No increased endothelial cell (EC) proliferation was detected in vivo, which was confirmed by in vitro administration to ECs. Immunohistochemistry and gene expression analyses indicated increased cellular stress and vascular endothelial growth factor (VEGF) expression in the liver, which may have contributed to the sinusoidal dilatation. A two-fold increase in the level of circulating VEGF was detected in the hamster at all dose levels, whereas no effect on VEGF was observed in patients treated with tesaglitazar. In conclusion, investigations have demonstrated that tesaglitazar does not produce hemangiosarcomas in hamster despite a slight effect on vascular morphology in the liver.
Assuntos
Alcanossulfonatos/toxicidade , Neoplasias Hepáticas Experimentais/induzido quimicamente , PPAR alfa/agonistas , PPAR gama/agonistas , Fenilpropionatos/toxicidade , Animais , Área Sob a Curva , Testes de Carcinogenicidade , Proliferação de Células/efeitos dos fármacos , Cricetinae , Feminino , Perfilação da Expressão Gênica , Hemangioma/induzido quimicamente , Hemangiossarcoma/induzido quimicamente , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Estatísticas não Paramétricas , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The dual peroxisome-proliferator-activated receptor (PPAR) α/γ agonist tesaglitazar has been shown to produce fibrosarcomas in rats. Here, the authors studied morphology, proliferation, differentiation, and inflammation markers in adipose tissue from rats exposed to 1, 3, or 10 µmol/kg tesaglitazar for 2 or 12 weeks, including recovery groups (12 weeks treatment followed by 12 weeks recovery), and 3 or 10 µmol/kg tesaglitazar for 24 weeks. Subcutaneous white and brown fat revealed reversible dose-related histopathological alterations and after 12 and 24 weeks developed areas of thickened skin (fatty lumps). There was a dose-dependent increase in proliferation of interstitial cells in white and brown fat as shown by increased mitotic index in all dose groups after 2 weeks. This was limited to the high dose after 12 and 24 weeks in white fat. Gene expression analyses showed that while tesaglitazar induced differentiation of adipose tissue characterized with a switch in cyclin D1 and D3 mRNA by 12 weeks, longer exposure at high doses reversed this differentiation concurrent with a reappearance of early adipocyte and inflammatory markers. These data suggest that sustained increased turnover of mesenchymal cells in adipose tissues, concomitant with onset of inflammation and fibrosis, drives development of fibrosarcomas in rats treated with tesaglitazar.
Assuntos
Tecido Adiposo/efeitos dos fármacos , Fibrossarcoma/induzido quimicamente , PPAR alfa/agonistas , PPAR gama/agonistas , Adipócitos , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Alcanossulfonatos/sangue , Alcanossulfonatos/metabolismo , Análise de Variância , Animais , Biomarcadores , Proliferação de Células , Fibrossarcoma/patologia , Expressão Gênica , Inflamação/induzido quimicamente , Masculino , Fenilpropionatos/sangue , Fenilpropionatos/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos WistarRESUMO
Despite the significance of mitochondrial ATP synthase for mammalian metabolism, the regulation of the amount of ATP synthase in mammalian systems is not understood. As brown adipose tissue mitochondria contain very low amounts of ATP synthase, relative to respiratory chain components, they constitute a physiological system that allows for examination of the control of ATP synthase assembly. To examine the role of the expression of the P1-isoform of the c-Fo subunit in the biogenesis of ATP synthase, we made transgenic mice that express the P1-c subunit isoform under the promoter of the brown adipose tissue-specific protein UCP1. In the resulting UCP1p1 transgenic mice, total P1-c subunit mRNA levels were increased; mRNA levels of other F1Fo-ATPase subunits were unchanged. In isolated brown-fat mitochondria, protein levels of the total c-Fo subunit were increased. Remarkably, protein levels of ATP synthase subunits that are part of the F1-ATPase complex were also increased, as was the entire Complex V. Increased ATPase and ATP synthase activities demonstrated an increased functional activity of the F1Fo-ATPase. Thus, the levels of the c-Fo subunit P1-isoform are crucial for defining the final content of the ATP synthase in brown adipose tissue. The level of c-Fo subunit may be a determining factor for F1Fo-ATPase assembly in all higher eukaryotes.
Assuntos
Tecido Adiposo Marrom/enzimologia , Isoenzimas/metabolismo , Mitocôndrias/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Animais , Sequência de Bases , Western Blotting , Primers do DNA , Efeito Fundador , Canais Iônicos/genética , Canais Iônicos/fisiologia , Camundongos , Camundongos Transgênicos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/fisiologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Desacopladora 1RESUMO
The development of the dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist tesaglitazar as an oral antidiabetic was recently discontinued. Here we present tumor data from a 2-year carcinogenicity study in rats given 0.3, 1, 3, and 10 micromol/kg tesaglitazar is presented with focus on the findings of subcutaneous fibrosarcomas. To investigate the mechanism for induction of fibrosarcomas, replicative DNA synthesis (immunohistochemical detection of BrdU-labeled cells) and expression of PPARgamma (immunohistochemistry and reverse transcription-polymerase chain reaction) in subcutaneous adipose tissues was assessed in rats administered 1 or 10 micromol/kg for 2 weeks or 3 months. Poorly differentiated subcutaneous mesenchymal sarcomas with a predominant spindle cell appearance occurred at the highest dose level of 10 micromol/kg in both sexes, and these tumors were diagnosed as fibrosarcomas. The 10-micromol/kg dose was at or above the maximum tolerated dose and caused considerable cardiovascular mortality. Tesaglitazar stimulated DNA synthesis mainly in subcutaneous interstitial mesenchymal cells. The percentage of BrdU-labeled interstitial cells was increased at 1 and 10 micromol/kg after 2 weeks. The increase in DNA synthesis was still significant at the end of the 12-week treatment at 10 mumol/kg, the dose producing fibrosarcoma. However, at 1 micromol/kg, a dose below the no-observed-effect level for fibrosarcoma, the level of DNA synthesis was similar to control levels at 12 weeks. Immunohistochemical analyses showed no detectable PPARgamma protein in the majority of BrdU-labeled interstitial mesenchymal cells in white and brown fat. This indicates that stimulation of DNA synthesis is not mediated via direct activation of PPARgamma in these cells. The results suggest that the induction of rat fibrosarcoma by tesaglitazar, at exposures 100-fold above the human therapeutic exposure, may involve proliferation of undifferentiated mesenchymal cells in subcutaneous tissues.
Assuntos
Alcanossulfonatos/farmacologia , DNA/biossíntese , Fibrossarcoma/induzido quimicamente , Hipoglicemiantes/farmacologia , Mesoderma/metabolismo , PPAR alfa/agonistas , PPAR gama/agonistas , Fenilpropionatos/farmacologia , Neoplasias Cutâneas/induzido quimicamente , Animais , Antimetabólitos , Bromodesoxiuridina , Colesterol/sangue , Replicação do DNA/efeitos dos fármacos , Feminino , Fibrossarcoma/patologia , Imuno-Histoquímica , Masculino , Mesoderma/efeitos dos fármacos , Microdissecção , Tamanho do Órgão/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , RNA/biossíntese , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/patologia , Triglicerídeos/sangueRESUMO
A significant amount of the fatty acids synthesized by the cytosolic enzyme complex fatty acid synthase (FAS) or taken up by the diet are further elongated into very long chain fatty acids (VLCFA) in a four-step reaction cycle by membrane-bound enzymes predominantly located in the endoplasmic reticulum. Members of the Elovl (elongation-of-very-long-chain-fatty acids) gene family encode for enzymes (elongases), which are believed to perform the first, regulatory, step (condensation) in the elongation cycle in mammals. The family of enzymes consists of at least six members in mouse and human, believed to carry out substrate-specific elongation with fatty acids of different lengths and degrees of unsaturation. The ability to synthesize VLCFA is a ubiquitous system found in different organs and cell types. However, VLCFAs seldom occur unesterified. Instead, they are joined either by an ester or amide linkage to a broad variety of different lipid species. VLCFA are most commonly found as building blocks in sphingolipids, although they are also important constituents of glycerophospholipids, triacylglycerols, sterol- and wax-esters. To generalize, the fatty acid elongases can be divided into two major groups: (a) enzymes which are suggested to be involved in the elongation of saturated and monounsaturated VLCFA (ELOVL1, 3 and 6) and (b) enzymes which are elongases of polyunsaturated fatty acids (PUFA) (ELOVL2, 4 and 5). All the elongases exhibit specific spatial and temporal expression. In this review, we will present and discuss the regulation of the mammalian fatty acid elongases and their potential role in lipid metabolism. We will consider both the biochemical functions of the proteins, as well as their role in a more physiological context.
Assuntos
Acetiltransferases/fisiologia , Metabolismo dos Lipídeos/fisiologia , Mamíferos/metabolismo , Acetiltransferases/genética , Animais , Elongases de Ácidos Graxos , Ácidos Graxos Insaturados/biossíntese , Regulação da Expressão Gênica , Proteínas de Membrana/fisiologiaRESUMO
During the recruitment process of brown adipose tissue, the mRNA level of the fatty acyl chain elongase Elovl3 is elevated more than 200-fold in cold-stressed mice. We have obtained Elovl3-ablated mice and report here that, although cold-acclimated Elovl3-ablated mice experienced an increased heat loss due to impaired skin barrier, they were unable to hyperrecruit their brown adipose tissue. Instead, they used muscle shivering in order to maintain body temperature. Lack of Elovl3 resulted in a transient decrease in the capacity to elongate saturated fatty acyl-CoAs into very long chain fatty acids, concomitantly with the occurrence of reduced levels of arachidic acid (C20:0) and behenic acid (C22:0) in brown adipose tissue during the initial cold stress. This effect on very long chain fatty acid synthesis could be illustrated as a decrease in the condensation activity of the elongation enzyme. In addition, warm-acclimated Elovl3-ablated mice showed diminished ability to accumulate fat and reduced metabolic capacity within the brown fat cells. This points to ELOVL3 as an important regulator of endogenous synthesis of saturated very long chain fatty acids and triglyceride formation in brown adipose tissue during the early phase of the tissue recruitment.
Assuntos
Tecido Adiposo/metabolismo , Proteínas de Membrana/fisiologia , Acetiltransferases , Tecido Adiposo Marrom/metabolismo , Animais , Temperatura Corporal , Peso Corporal , Cromatografia Gasosa , Temperatura Baixa , Cruzamentos Genéticos , Ácidos Eicosanoicos/química , Eletromiografia , Elongases de Ácidos Graxos , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Feminino , Lipídeos/química , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/metabolismo , Consumo de Oxigênio , RNA/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Triglicerídeos/metabolismoRESUMO
Very little is known about the in vivo regulation of mammalian fatty acid chain elongation enzymes as well as the role of specific fatty acid chain length in cellular responses and developmental processes. Here, we report that the Elovl3 gene product, which belongs to a highly conserved family of microsomal enzymes involved in the formation of very long chain fatty acids, revealed a distinct expression in the skin that was restricted to the sebaceous glands and the epithelial cells of the hair follicles. By disruption of the Elovl3 gene by homologous recombination in mouse, we show that ELOVL3 participates in the formation of specific neutral lipids that are necessary for the function of the skin. The Elovl3-ablated mice displayed a sparse hair coat, the pilosebaceous system was hyperplastic, and the hair lipid content was disturbed with exceptionally high levels of eicosenoic acid (20:1). This was most prominent within the triglyceride fraction where fatty acids longer than 20 carbon atoms were almost undetectable. A functional consequence of this is that Elovl3-ablated mice exhibited a severe defect in water repulsion and increased trans-epidermal water loss.