RESUMO
The protein encoded by the G0/G1 switch gene 2 (G0S2) is a potent inhibitor of adipose triglyceride lipase (ATGL) and thus an important regulator of intracellular lipolysis. Since dysfunction of lipolysis is associated with metabolic diseases including diabetes and obesity, inhibition of ATGL is considered a therapeutic strategy. G0S2 interacts with ATGL's patatin-domain to mediate non-competitive inhibition, however atomic details of the inhibition mechanism are incompletely understood. Sequences of G0S2 from higher organisms show a highly conserved N-terminal part, including a hydrophobic region covering amino acids 27 to 42. We show that predicted G0S2 orthologs from platypus, chicken and Japanese rice-fish are able to inhibit human and mouse ATGL, emphasizing the contribution of conserved amino acid to ATGL inhibition. Our site directed mutagenesis and truncation studies give insights in the protein-protein interaction on a per-residue level. We determine that the minimal sequence required for ATGL inhibition ranges from amino acids 20 to 44. Residues Y27, V28, G30, A34 G37, V39 or L42 within this sequence play a substantial role in ATGL inhibition. Furthermore, we show that unspecific interactions of the N-terminal part (amino acids 20-27) of the minimal sequence facilitate the interaction to ATGL. Our studies also demonstrate that full-length G0S2 shows higher tolerance to specific single amino acid exchanges in the hydrophobic region due to the stronger contributions of unspecific interactions. However, exchanges of more than one amino-acid in the hydrophobic region also result in the loss of function as ATGL inhibitor even in the full-length protein.
Assuntos
LipóliseRESUMO
AIMS/HYPOTHESIS: The enzyme hormone-sensitive lipase (HSL) is produced and is active in pancreatic beta cells. Because lipids are known to play a crucial role in normal control of insulin release and in the deterioration of beta cell function, as observed in type 2 diabetes, actions of HSL in beta cells may be critical. This notion has been addressed in different lines of HSL knockout mice with contradictory results. METHODS: To resolve this, we created a transgenic mouse lacking HSL specifically in beta cells, and characterised this model with regard to glucose metabolism and insulin secretion, using both in vivo and in vitro methods. RESULTS: We found that fasting basal plasma glucose levels were significantly elevated in mice lacking HSL in beta cells. An IVGTT at 12 weeks revealed a blunting of the initial insulin response to glucose with delayed elimination of the sugar. Additionally, arginine-stimulated insulin secretion was markedly diminished in vivo. Investigation of the exocytotic response in single HSL-deficient beta cells showed an impaired response to depolarisation of the plasma membrane. Beta cell mass and islet insulin content were increased, suggesting a compensatory mechanism, by which beta cells lacking HSL strive to maintain normoglycaemia. CONCLUSIONS/INTERPRETATION: Based on these results, we suggest that HSL, which is located in close proximity of the secretory granules, may serve as provider of a lipid-derived signal essential for normal insulin secretion.
Assuntos
Hiperglicemia/etiologia , Células Secretoras de Insulina/enzimologia , Insulina/metabolismo , Esterol Esterase/deficiência , Esterol Esterase/genética , Tecido Adiposo/enzimologia , Animais , Área Sob a Curva , Glicemia/metabolismo , Exocitose/genética , Éxons , Teste de Tolerância a Glucose , Hiperglicemia/sangue , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Vesículas Secretórias/enzimologiaRESUMO
The chronic and acute effects of different types of dietary fat on postprandial lipoprotein metabolism were studied in eight normolipidemic subjects. Each person was placed for 25 d on each of three isocaloric diets: a saturated fat (SFA), a w-6 polyunsaturated fat (w-6 PUFA) and a w-3 polyunsaturated fat (w-3 PUFA) diet. Two vitamin A-fat loading tests were done on each diet. The concentrations in total plasma and chylomicron (Sf greater than 1,000) and nonchylomicron (Sf less than 1,000) fractions of retinyl palmitate (RP) were measured for 12 h postprandially. Compared with the SFA diet, the w-6 PUFA diet reduced chylomicron and nonchylomicron RP levels 56 and 38%, respectively, and the w-3 PUFA diet reduced these levels 67 and 53%, respectively. On further analysis, the main determinant of postprandial lipoprotein levels was the type of fat that was chronically fed, which appeared to mediate its effect by changing the concentration of the endogenous competitor for the system that catabolizes triglyeride-rich lipoproteins. However, there was a significant effect of the acute dietary fat load, which appeared to be due to a differential susceptibility to lipolysis of chylomicrons produced by SFA as opposed to PUFA fat loads. The levels of postprandial lipoproteins are determined by the interaction of these chronic and acute effects.
Assuntos
Gorduras na Dieta/farmacologia , Ácidos Graxos Insaturados/farmacologia , Lipoproteínas/sangue , Adulto , Quilomícrons/análise , Gorduras na Dieta/administração & dosagem , Diterpenos , Ácidos Graxos Insaturados/administração & dosagem , Hemoglobinas Glicadas/análise , Humanos , Insulina/sangue , Lipase/sangue , Lipólise , Masculino , Contagem de Plaquetas , Valores de Referência , Ésteres de Retinil , Vitamina A/análogos & derivados , Vitamina A/sangue , Vitamina A/farmacocinéticaRESUMO
The composition of lipoproteins in the plasma of patients with LCAT deficiency (LCAT-D) is grossly altered due to the lack of cholesteryl esters which form the core of normal lipoproteins. When plasma from LCAT-D patients and their relatives was examined we found that nine heterozygotes had plasma Lp(a) levels of 2-13 mg/dl whereas none of 11 affected homozygous individuals from different families contained detectable amounts of Lp(a) in their plasma. Therefore, the binding of apo(a) to LDL density particles was studied in vitro using LDL density fractions prepared from patients, and recombinant apo(a) [r-apo(a)], which was expressed and secreted by transfected COS-7 cells. The LDL from heterozygotes were chemically indistinguishable from normal LDL and homogeneous with regard to morphology, whereas the crude LDL floating fraction from homozygotes consisted of a heterogeneous mixture of large vesicles, and small spheres resembling normal LDL. The LDL density fraction from the LCAT-D patient lacked almost completely cholesteryl esters. Incubation of LCAT-D plasma with active LCAT caused a substantial augmentation of the original subfraction which morphologically resembled normal LDL. Using r-apo(a) and normal LDL or LDL of heterozygous individuals, apoB:r-apo(a) complexes were formed when incubated at 37 degrees C in vitro for 20 h. In contrast, the total LDL floating fraction from a homozygous LCAT-D patient failed to form apoB:r-apo(a) complexes. After treatment with active LCAT, a significant apoB:r-apo(a) association was observed with LCAT-D LDL-density particles. Our data emphasize the importance of the integrity of LDL structure and composition for the formation of Lp(a). In addition, we demonstrate that the absence of LCAT activity has a fundamental impact on the regulation of plasma Lp(a) levels.
Assuntos
Apolipoproteínas A/biossíntese , Deficiência da Lecitina Colesterol Aciltransferase/metabolismo , Lipoproteínas LDL/biossíntese , Apolipoproteínas A/sangue , Apolipoproteínas A/metabolismo , Apolipoproteínas B/metabolismo , Áustria/epidemiologia , Feminino , Homozigoto , Humanos , Deficiência da Lecitina Colesterol Aciltransferase/epidemiologia , Lipoproteínas LDL/química , Lipoproteínas LDL/ultraestrutura , Masculino , Linhagem , Ligação ProteicaRESUMO
Hypertriglyceridemia is common in the general population, but its mechanism is largely unknown. In previous work human apo CIII transgenic (HuCIIITg) mice were found to have elevated triglyceride levels. In this report, the mechanism for the hypertriglyceridemia was studied. Two different HuCIIITg mouse lines were used: a low expressor line with serum triglycerides of approximately 280 mg/dl, and a high expressor line with serum triglycerides of approximately 1,000 mg/dl. Elevated triglycerides were mainly in VLDL. VLDL particles were 1.5 times more triglyceride-rich in high expressor mice than in controls. The total amount of apo CIII (human and mouse) per VLDL particle was 2 and 2.5 times the normal amount in low and high expressors, respectively. Mouse apo E was decreased by 35 and 77% in low and high expressor mice, respectively. Under electron microscopy, VLDL particles from low and high expressor mice were found to have a larger mean diameter, 55.2 +/- 16.6 and 58.2 +/- 17.8 nm, respectively, compared with 51.0 +/- 13.4 nm from control mice. In in vivo studies, radiolabeled VLDL fractional catabolic rate (FCR) was reduced in low and high expressor mice to 2.58 and 0.77 pools/h, respectively, compared with 7.67 pools/h in controls, with no significant differences in the VLDL production rates. In an attempt to explain the reduced VLDL FCR in transgenic mice, tissue lipoprotein lipase (LPL) activity was determined in control and high expressor mice and no differences were observed. Also, VLDLs obtained from control and high expressor mice were found to be equally good substrates for purified LPL. Thus excess apo CIII in HuCIIITg mice does not cause reduced VLDL FCR by suppressing the amount of extractable LPL in tissues or making HuCIIITg VLDL a bad substrate for LPL. Tissue uptake of VLDL was studied in hepatoma cell cultures, and VLDL from transgenic mice was found to be taken up much more slowly than control VLDL (P < 0.0001), indicating that HuCIIITg VLDL is not well recognized by lipoprotein receptors. Additional in vivo studies with Triton-treated mice showed increased VLDL triglyceride, but not apo B, production in the HuCIIITg mice compared with controls. Tissue culture studies with primary hepatocytes showed a modest increase in triglyceride, but not apo B or total protein, secretion in high expressor mice compared with controls. In summary, hypertriglyceridemia in HuCIIITg mice appears to result primarily from decreased tissue uptake of triglyceride-rich particles from the circulation, which is most likely due to increased apo CIII and decreased apo E on VLDL particles. the HuCIIITg mouse appears to be a suitable animal model of primary familial hypertriglyceridemia, and these studies suggest a possible mechanism for this common lipoprotein disorder.
Assuntos
Apolipoproteínas C/metabolismo , Apolipoproteínas E/metabolismo , Hipertrigliceridemia/etiologia , Lipoproteínas VLDL/metabolismo , Animais , Apolipoproteína C-III , Apolipoproteínas C/genética , Células Cultivadas , Feminino , Humanos , Lipase Lipoproteica/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Triglicerídeos/metabolismoRESUMO
Lipoprotein lipase (LPL)-deficient mice have been created by gene targeting in embryonic stem cells. At birth, homozygous knockout pups have threefold higher triglycerides and sevenfold higher VLDL cholesterol levels than controls. When permitted to suckle, LPL-deficient mice become pale, then cyanotic, and finally die at approximately 18 h of age. Before death, triglyceride levels are severely elevated (15,087 +/- 3,805 vs 188 +/- 71 mg/dl in controls). Capillaries in tissues of homozygous knockout mice are engorged with chylomicrons. This is especially significant in the lung where marginated chylomicrons prevent red cell contact with the endothelium, a phenomenon which is presumably the cause of cyanosis and death in these mice. Homozygous knockout mice also have diminished adipose tissue stores as well as decreased intracellular fat droplets. By crossbreeding with transgenic mice expressing human LPL driven by a muscle-specific promoter, mouse lines were generated that express LPL exclusively in muscle but not in any other tissue. This tissue-specific LPL expression rescued the LPL knockout mice and normalized their lipoprotein pattern. This supports the contention that hypertriglyceridemia caused the death of these mice and that LPL expression in a single tissue was sufficient for rescue. Heterozygous LPL knockout mice survive to adulthood and have mild hypertriglyceridemia, with 1.5-2-fold elevated triglyceride levels compared with controls in both the fed and fasted states on chow, Western-type, or 10% sucrose diets. In vivo turnover studies revealed that heterozygous knockout mice had impaired VLDL clearance (fractional catabolic rate) but no increase in transport rate. In summary, total LPL deficiency in the mouse prevents triglyceride removal from plasma, causing death in the neonatal period, and expression of LPL in a single tissue alleviates this problem. Furthermore, half-normal levels of LPL cause a decrease in VLDL fractional catabolic rate and mild hypertriglyceridemia, implying that partial LPL deficiency has physiological consequences.
Assuntos
Hipertrigliceridemia/fisiopatologia , Lipase Lipoproteica/deficiência , Lipase Lipoproteica/genética , Lipoproteínas HDL/sangue , Triglicerídeos/sangue , Tecido Adiposo/patologia , Animais , Animais Recém-Nascidos , Southern Blotting , Composição Corporal , Colesterol/sangue , DNA/análise , Morte , Feminino , Genótipo , Heterozigoto , Hipertrigliceridemia/genética , Hipertrigliceridemia/patologia , Absorção Intestinal , Lipoproteínas LDL/sangue , Fígado/patologia , Fígado/ultraestrutura , Camundongos , Camundongos Knockout , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Gravidez , Células-Tronco , Vitamina A/metabolismoRESUMO
In extrahepatic tissues lipoprotein lipase (LPL) hydrolyzes triglycerides thereby generating FFA for tissue uptake and metabolism. To study the effects of increased FFA uptake in muscle tissue, transgenic mouse lines were generated with a human LPL minigene driven by the promoter of the muscle creatine kinase gene. In these mice human LPL was expressed in skeletal muscle and cardiac muscle, but not in other tissues. In proportion to the level of LPL overexpression, decreased plasma triglyceride levels, elevated FFA uptake by muscle tissue, weight loss, and premature death were observed in three independent transgenic mouse lines. The animals developed a severe myopathy characterized by muscle fiber degeneration, fiber atrophy, glycogen storage, and extensive proliferation of mitochondria and peroxisomes. This degree of proliferation suggests that FFA play an important role in the biogenesis of these organelles. Our experiments indicate that LPL is rate limiting for the supply of muscle tissue with triglyceride-derived FFA. Improper regulation of muscle LPL can lead to major pathological changes and may be important in the pathogenesis of some human myopathies. Muscle-specific LPL transgenic mouse lines will serve as a useful animal model for the investigation of myopathies and the biogenesis of mitochondria and peroxisomes.
Assuntos
Ácidos Graxos não Esterificados/metabolismo , Lipase Lipoproteica/genética , Microcorpos/patologia , Mitocôndrias Musculares/patologia , Miopatias Mitocondriais/etiologia , Proteínas Musculares/genética , Proteínas Recombinantes de Fusão/biossíntese , Animais , Creatina Quinase/genética , Indução Enzimática , Genes Sintéticos , Glucose/metabolismo , Humanos , Lipólise , Lipase Lipoproteica/biossíntese , Longevidade , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Miopatias Mitocondriais/enzimologia , Miopatias Mitocondriais/genética , Proteínas Musculares/biossíntese , Músculos/metabolismo , Músculos/patologia , Especificidade de Órgãos , Regiões Promotoras Genéticas , Triglicerídeos/sangue , Redução de PesoRESUMO
Lipoprotein lipase (LPL) is synthesized primarily in muscle and adipose tissue and by hydrolyzing triglycerides in chylomicrons and very low density lipoprotein allows uptake of the resultant free fatty acids by these tissues. This report describes the cloning of the mouse LPL gene from which probes were derived to study the regulation of LPL synthesis in the 3T3-L1 adipocyte cell culture system. Preconfluent 3T3-L1 preadipocytes had very small amounts of LPL mRNA (less than 1 pg/micrograms of RNA). At confluency, LPL mRNA levels increased to 5 to 15 pg/micrograms of RNA. After insulin and dexamethasone were added, LPL activity and mRNA levels rose in parallel. Peak mRNA levels were reached within 4 to 10 days, achieving LPL mRNA concentrations of 150 to 500 pg/micrograms of RNA. This represents a 15- to 50-fold increase over confluent cells. Two cytokines known to diminish adipose tissue LPL activity were studied to see how their effects were regulated. Recombinant human cachectin/tumor necrosis factor diminished both LPL activity and LPL mRNA levels. The effect on LPL activity compared with mRNA levels was quicker, at a lower dose, and more complete (95 versus 75% maximum effect). The effect of recombinant human cachectin tumor necrosis factor on LPL mRNA levels was shown by nuclear run-on experiments to be exerted transcriptionally. It was also independent of new protein synthesis. Recombinant human interleukin-1 alpha diminished only LPL activity but not mRNA levels. This study suggests that during times of stress, cytokines secreted by activated macrophages can alter energy balance by affecting transcriptional and posttranscriptional processes in adipocytes.
Assuntos
Tecido Adiposo/metabolismo , Interleucina-1/farmacologia , Lipase Lipoproteica/genética , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Tecido Adiposo/citologia , Tecido Adiposo/efeitos dos fármacos , Animais , Sequência de Bases , Células Cultivadas , Clonagem Molecular , Repressão Enzimática , Camundongos , Dados de Sequência MolecularRESUMO
Lipoprotein lipase (LPL) is an important enzyme in lipid and energy metabolism of all vertebrates. Measurement of its activity in human postheparin plasma has become a standard procedure for diagnosis of Type I hyperlipoproteinemia and other types of hypertriglyceridemias. This paper presents a rapid and simple purification procedure for human lipoprotein lipase and the production of specific polyclonal antibodies. In the isolation procedure, the fat moiety of human milk obtained by centrifugation was delipidated and a buffer-extractable fraction chromatographed sequentially on heparin-Sepharose and phenyl-Sepharose. This three-step procedure provides a high yield of apparently pure LPL with very high specific activity against radiolabeled triacylglycerol substrates. The apparent molecular weight of LPL on SDS-PAGE was 60 kDa. Amino acid analysis and NH2-terminal sequencing proved the identity and the apparent homogeneity of the isolated enzyme. alpha-Lactoferrin and antithrombin III, common contaminants in earlier isolation procedures, were not detectable immunologically. Purified LPL was used to produce in the rabbit a specific polyclonal antiserum that inhibited LPL activity from human postheparin plasma and other tissues. In postheparin plasma from normal individuals, anti-LPL IgG was used in Western blotting to show LPL protein. In preheparin plasma, or in certain patients with Type I hyperlipoproteinemia, no specific signal was detected. The improved purification procedure presented here allows the rapid isolation of human LPL and production of antibodies to the protein, both of which will greatly facilitate future studies of this important enzyme.
Assuntos
Lipase Lipoproteica/isolamento & purificação , Leite Humano/enzimologia , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Western Blotting , Bovinos , Cromatografia em Agarose , Eletroforese em Gel de Poliacrilamida , Cobaias , Humanos , Lipase Lipoproteica/sangue , Lipase Lipoproteica/imunologia , Camundongos , Dados de Sequência Molecular , CoelhosRESUMO
The conversion of pig high-density lipoproteins (HDL) (mainly HDL3) to fractions of lower densities was studied by incubating pig plasma for 24 h at 37 degrees C in the presence and absence of lipoprotein lipase from bovine milk, lecithin:cholesterol acyltransferase, cholesteryl ester transfer protein and triacylglycerol-rich particles (very-low-density lipoproteins (VLDL) or Intralipid). The results can be summarized as follows. In the presence of lipoprotein lipase and at a VLDL/HDL mass ratio of 2, the F-1.210 of pig HDL was shifted from 3.3 to 4.2, which is characteristic for human HDL2. This shift was caused by the excessive increase in the free fatty acid content in HDL. If 50 g/l of bovine serum albumin were added prior to incubation, the flotation rate of HDL remained in the HDL2a region. If lecithin:cholesterol acyltransferase was active in fasting pig plasma during incubation, we observed only a negligible increase of F-1.210 in HDL. If pig lipoproteins were incubated with human lipoprotein-free serum as a source of cholesteryl ester transfer activity, a slight increase in the flotation rate of HDL was observed, which was amplified in the presence of active lecithin:cholesterol acyltransferase. Pig HDL was converted to a fraction with F-1.210 of 4.2, which is typical for human HDL2, only if active lecithin:cholesterol acyltransferase, cholesteryl ester transfer protein and triacylglycerol-rich particles were present in the incubation mixture. From our results we also concluded that apolipoprotein A-II plays no role in the HDL2 formation.
Assuntos
Lipoproteínas HDL/sangue , Animais , Humanos , Cinética , Lipase Lipoproteica/metabolismo , Lipoproteínas HDL/isolamento & purificação , Lipoproteínas VLDL/sangue , Lipoproteínas VLDL/isolamento & purificação , Fosfatidilcolina-Esterol O-Aciltransferase/sangue , Especificidade da Espécie , SuínosRESUMO
In order to study the factors responsible for the formation of high-density lipoprotein subfraction-2 (HDL-2), very-low-density lipoproteins (VLDL) and HDL-3 were mixed and incubated with purified bovine milk lipoprotein lipase, human serum lecithin:cholesterol acyltransferase, cholesteryl ester transfer protein and mixtures thereof. The results can be summarized as follows: Incubation of HDL-3 and VLDL for 24 h at 37 degrees C without enzymes did not cause any significant change in the protein:lipid ratio or in the flotation constant of the HDL. Cholesteryl ester transfer protein treatment caused only an exchange of part of the HDL cholesteryl esters with VLDL triacylglycerols. Lipoprotein lipase caused a slight shift of HDL-hydrated density to lower values; HDL-2b, however, was not formed. Incubation of HDL-3 and VLDL with lecithin:cholesterol acyltransferase or mixtures of lecithin:cholesterol acyltransferase and lipoprotein lipase reduced the HDL-protein:lipid ratio and increased the HDL-flotation rate. The newly formed HDL resembled that of native HDL-2a. The incubation of HDL-3 and VLDL with lecithin:cholesterol acyltransferase and cholesteryl ester transfer protein caused a shift of the HDL-3 into an HDL-2b-like fraction. Particles resembling HDL-2b in the analytical ultracentrifuge were also formed if VLDL + HDL-3 were incubated with lipoprotein lipase or lipoprotein lipase + cholesteryl ester transfer protein in a medium containing low amounts of albumin, insufficient for binding all liberated fatty acids during hydrolysis. The incubation of mixtures of HDL-3 and chylomicrons enriched with apoAI in the presence of lecithin:cholesterol acyltransferase and cholesteryl ester transfer protein caused the formation of HDL-2-like particles which resembled those of native HDL-2 also with respect to the apoAI/AII ratio.
Assuntos
Proteínas de Transporte/metabolismo , Glicoproteínas , Lipoproteínas HDL/metabolismo , Lipoproteínas VLDL/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Fenômenos Químicos , Físico-Química , Proteínas de Transferência de Ésteres de Colesterol , Eletroforese em Gel de Poliacrilamida , Humanos , Lipoproteínas HDL2 , Lipoproteínas HDL3RESUMO
Macrophages are intimately involved in the pathogenesis of atherosclerotic diseases. A key feature of this process is their uptake of various lipoproteins and subsequent transformation to foam cells. Since lipoprotein lipase (LPL) is believed to play a role in foam cell formation, we investigated if endogenously produced proteoglycans (PGs) affect the release of this enzyme from macrophages. The human leukaemic cell line THP-1 which differentiates into macrophages by treatment with phorbol ester (phorbol 12-myristate 13-acetate) served as a model. The differentiation of THP-1 macrophages promoted the release of PGs into the cell medium which caused the detachment of LPL activity from the cell surface, and prevented LPL re-uptake and inactivation. These PGs were mainly composed of chondroitin sulfate type and exerted a heparin-like effect on LPL release. LPL is known to increase the cell association of lipoproteins by the well known bridging function. Exogenous bovine LPL at a concentration of 1 microg/ml enhanced low density lipoprotein (LDL)-binding 10-fold. Endogenously produced PGs reduced LPL-mediated binding of LDL. It is proposed that the differentiation-dependent increase in the release of PGs interferes with binding of LPL and reduces lipoprotein-binding to macrophages.
Assuntos
Glicosaminoglicanos/metabolismo , Lipase Lipoproteica/metabolismo , Lipoproteínas/metabolismo , Macrófagos/metabolismo , Proteoglicanas/metabolismo , Arteriosclerose/metabolismo , Linhagem Celular , Sulfatos de Condroitina/metabolismo , Meios de Cultivo Condicionados , Dermatan Sulfato/metabolismo , Glicosaminoglicanos/biossíntese , Heparina/farmacologia , Humanos , Lipase Lipoproteica/farmacologia , Lipoproteínas LDL/metabolismo , Proteoglicanas/biossíntese , Proteoglicanas/químicaRESUMO
BACKGROUND: The major source of nitric oxide (NO) in the heart is the constitutive form of NO synthases (eNOS, NOS III) that is expressed in vascular endothelium and cardiac myocytes. NO mediates endothelium-dependent vasodilation and may modulate cardiac function. We examined the role of NO in hearts from transgenic (TG) mice overexpressing eNOS exclusively in cardiac myocytes. METHODS AND RESULTS: Three independent TG lines with varying levels of NOS activity were selected, and the hearts were isolated and retrogradely perfused at constant flow. We found that NO is positively inotropic in spontaneously beating hearts from wild-type (WT) mice, whereas hearts overexpressing eNOS had reduced basal contractility that was partially reversed by NOS blockade. Heart rate was not altered. Acetylcholine (10 to 1000 nmol/L) increased contractility in unstimulated hearts and decreased contractility after beta-adrenergic stimulation with norepinephrine, and these responses were identical in WT and TG hearts. Finally, resting systolic intracellular calcium (Ca(2+)(i)) tended to be lower in TG than in WT hearts, and the beat-to-beat responsiveness to Ca(2+)(i) was reduced in hearts with eNOS overexpression. CONCLUSIONS: High levels of endogenous myocyte-derived NO blunt myofilament Ca(2+) sensitivity. The similar effects of acetylcholine on contractility and heart rate, as well as the identical basal intrinsic heart rate in WT and TG hearts, provide a solid argument against NO as an important modulator of neurohormonal control of myocardial function.
Assuntos
Frequência Cardíaca/fisiologia , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Óxido Nítrico Sintase/metabolismo , Acetilcolina/farmacologia , Animais , Cálcio/metabolismo , Cálcio/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Feminino , Regulação Enzimológica da Expressão Gênica , Genótipo , Frequência Cardíaca/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Contração Miocárdica/efeitos dos fármacos , Miocárdio/enzimologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Norepinefrina/farmacologia , Vasoconstritores , Vasodilatadores/farmacologia , Função VentricularRESUMO
In patients with type 2 diabetes, a strong correlation between accumulation of intramuscular triclycerides (TGs) and insulin resistance has been found. The aim of the present study was to determine whether there is a causal relation between intramuscular TG accumulation and insulin sensitivity. Therefore, in mice with muscle-specific overexpression of human lipoprotein lipase (LPL) and control mice, muscle TG content was measured in combination with glucose uptake in vivo, under hyperinsulinemic-euglycemic conditions. Overexpression of LPL in muscle resulted in accumulation of TGs in skeletal muscle (85.5 +/- 33.3 vs. 25.7 +/- 23.1 micromol/g tissue in LPL and control mice, respectively; P < 0.05). During the hyperinsulinemic clamp study, there were no differences in plasma glucose, insulin, and FFA concentrations between the two groups. Moreover, whole-body, as well as skeletal muscle, insulin-mediated glucose uptake did not differ between LPL-overexpressing and wild-type mice. Surprisingly, whole-body glucose oxidation was decreased by approximately 60% (P < 0.05), whereas nonoxidative glucose disposal was increased by approximately 50% (P < 0.05) in LPL-overexpressing versus control mice. In conclusion, overexpression of human LPL in muscle increases intramuscular TG accumulation, but does not affect whole-body or muscle-specific insulin-mediated uptake, findings that argue against a simple causal relation between intramuscular TG content and insulin resistance.
Assuntos
Glucose/metabolismo , Insulina/fisiologia , Lipase Lipoproteica/metabolismo , Músculo Esquelético/enzimologia , Triglicerídeos/metabolismo , Animais , Sangue/metabolismo , Desoxiglucose/farmacocinética , Glicogênio/biossíntese , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Valores de ReferênciaRESUMO
The nonenzymatic glycation of LDL is a naturally occurring chemical modification of apolipoprotein (apo)-B lysine residues by glucose. Once glycated, LDL is only poorly recognized by lipoprotein receptors including the LDL receptor (LDL-R), the LDL-R-related protein (LRP), and scavenger receptors. Glycated LDL (gLDL) is a preferred target for oxidative modifications. Additionally, its presence initiates different processes that can be considered "proatherogenic." Thus, LDL glycation might contribute to the increased atherosclerotic risk of patients with diabetes and familial hypercholesterolemia. Here we investigate whether lipoprotein lipase (LPL) can mediate the cellular uptake of gLDL. The addition of exogenous LPL to the culture medium of human skin fibroblasts, porcine aortic endothelial cells, and mouse peritoneal macrophages enhanced the binding, uptake, and degradation of gLDL markedly, and the relative effect of LPL on lipoprotein uptake increased with the degree of apoB glycation. The efficient uptake of gLDL by LDL-R-deficient fibroblasts and LRP-deficient Chinese hamster ovary cells in the presence of LPL suggested a mechanism that was independent of the LDL-R and LRP. In macrophages, the uptake of gLDL was also correlated with their ability to produce LPL endogenously. Mouse peritoneal macrophages from genetically modified mice, which lacked LPL, exhibited a 75% reduction of gLDL uptake compared with normal macrophages. The LPL-mediated effect required the association of the enzyme with cell surface glycosaminoglycans but was independent of its enzymatic activity. The uptake of gLDL in different cell types by an LPL-mediated process might have important implications for the cellular response after gLDL exposure as well as the removal of gLDL from the circulation.
Assuntos
Endotélio Vascular/metabolismo , Fibroblastos/metabolismo , Lipase Lipoproteica/metabolismo , Lipoproteínas LDL/farmacocinética , Macrófagos/metabolismo , Acetilação , Animais , Células CHO , Linhagem Celular , Cricetinae , Produtos Finais de Glicação Avançada , Humanos , Luz , Receptores de LDL/metabolismo , Espalhamento de Radiação , Regulação para CimaRESUMO
Lipoprotein lipase (LPL) synthesis by macrophages is upregulated in early atherogenesis, implicating the possible involvement of LPL in plaque formation. However, it is still unclear whether macrophage-derived LPL displays a proatherosclerotic or an antiatherosclerotic role in atherosclerotic lesion development. In this study, the role of macrophage-derived LPL on lipid metabolism and atherosclerosis was assessed in vivo by transplantation of LPL-deficient (LPL-/-) and wild-type (LPL+/+) bone marrow into C57BL/6 mice. Eight weeks after bone marrow transplantation (BMT), serum cholesterol levels in LPL-/--->C57BL/6 mice were reduced by 8% compared with those in LPL+/+-->C57BL/6 mice (P:<0.05, n=16), whereas triglycerides were increased by 33% (P:<0.05, n=16). Feeding the mice a high-cholesterol diet increased serum cholesterol levels in LPL-/--->C57BL/6 and LPL+/+-->C57BL/6 mice 5-fold and 9-fold, respectively, resulting in a difference of approximately 50% (P:<0. 01) after 3 months on the diet. No effects on triglyceride levels were observed under these conditions. Furthermore, serum apolipoprotein E levels were reduced by 50% in the LPL-/--->C57BL/6 mice compared with controls under both dietary conditions. After 3 months on a high-cholesterol diet, the atherosclerotic lesion area in LPL-/--->C57BL/6 mice was reduced by 52% compared with controls. It can be concluded that macrophage-derived LPL plays a significant role in the regulation of serum cholesterol, apolipoprotein E, and atherogenesis, suggesting that specific blockade of macrophage LPL production may be beneficial for decreasing atherosclerotic lesion development.
Assuntos
Arteriosclerose/metabolismo , Lipase Lipoproteica/metabolismo , Lipoproteínas/metabolismo , Macrófagos/enzimologia , Animais , Apolipoproteínas E/sangue , Arteriosclerose/sangue , Arteriosclerose/enzimologia , Transplante de Medula Óssea , Colesterol/sangue , Colesterol na Dieta , Feminino , Humanos , Iodo/metabolismo , Lipoproteínas VLDL/metabolismo , Testes de Função Hepática , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Triglicerídeos/sangueRESUMO
In a comprehensive study the concentrations of plasma lipids and lipo- and apolipoproteins were measured in 24 nonpregnant women (control) and longitudinally in 42 women throughout gestation and postpartum. The results were correlated with hCG, 17 beta-estradiol (E2), progesterone (PG), human placental lactogen (hPL), and insulin levels by time series analysis. Insulin concentrations were constant until week 25 and increased thereafter. Plasma E2, PG, and hPL as well as plasma lipid levels rose continuously during gestation. Apolipoproteins AI, AII, and B concentrations increased until weeks 25, 28, and 32, respectively, and remained constant until term. Low density lipoprotein cholesterol reached maximum levels at week 36. High density lipoprotein cholesterol exhibited a triphasic behavior, with maximum levels at week 25, a fall until week 32, and maintenance of the level until term. Time series analysis revealed positive correlations with E2, PG, and hPL. These results provide evidence that apoprotein concentrations undergo pronounced serial changes during gestation, which in part might be due to the effect of E2. Furthermore, the importance of hPL as a determinant of the plasma levels of total and free cholesterol, triglycerides, and phospholipids is now documented.
Assuntos
Apolipoproteínas/sangue , Hormônios Esteroides Gonadais/sangue , Lipídeos/sangue , Hormônios Placentários/sangue , Período Pós-Parto/sangue , Gravidez/sangue , Adulto , Gonadotropina Coriônica/sangue , Estradiol/sangue , Feminino , Humanos , Insulina/sangue , Lipoproteínas/sangue , Lactogênio Placentário/sangue , Progesterona/sangueRESUMO
A transgenic mouse model for peroxisomal and mitochondrial induction caused by increased uptake of fatty acids in muscle was established. Transgenic mouse lines were generated using a human lipoprotein lipase (LPL) mini gene (3-20 copies) driven by the promoter of the muscle creatine kinase gene. Expression of human LPL was only observed in skeletal and cardiac muscle. In proportion to the level of LPL overexpression increased LPL activity in skeletal (up to 24-fold) and cardiac (up to three-fold) muscle, decreased plasma triglyceride levels, elevated free fatty acid (FFA) uptake by muscle tissue, weight loss (due to a reduction in muscle mass as well as adipose tissue mass) and premature death were observed. A remarkable increase in the number of mitochondria and peroxisomes was detected using oxide-electron microscopy. Proliferation of mitochondria and peroxisomes was confirmed by a dose-dependent increase of marker enzyme activity (succinate-dehydrogenase and catalase). In addition, peroxisomal acyl-CoAse enzyme protein was markedly elevated whereas mRNA was increased only up to two-fold. No changes in peroxisomal proliferator activated receptor alpha mRNA was found. This degree of proliferation and enzyme activity of mitochondria and peroxisomes suggests that FFA play an important role in the induction of these organelles. In addition, myopathy characterized by excessive glycogen storage, muscle fiber degeneration, and fiber atrophy with centralization of nuclei, mimicking several forms of human myopathies was noted. Our results imply that improper regulation of muscle LPL leading to increased fatty acid levels in muscle can cause severe pathological changes. This effect may be important in the pathogenesis of human myopathies. We conclude that these transgenic mouse lines could serve as a useful animal model for the investigation of myopathies and the effects of fatty acids on the induction of mitochondria and peroxisomes.
Assuntos
Ácidos Graxos não Esterificados/metabolismo , Lipase Lipoproteica/metabolismo , Microcorpos/enzimologia , Músculos/metabolismo , Animais , Composição Corporal , Peso Corporal , Citoplasma/metabolismo , Indução Enzimática , Humanos , Metabolismo dos Lipídeos , Camundongos , Camundongos Transgênicos , Microcorpos/ultraestrutura , Microscopia Eletrônica , Mitocôndrias Musculares/metabolismo , Proteínas Musculares/metabolismo , Músculos/anatomia & histologiaRESUMO
The effect of bezafibrate on plasma lipids, lipoproteins, apolipoproteins AI, AII and B, and LCAT activity was investigated in 16 hyperlipidemic, non-insulin-dependent diabetes, who were treated for 8 weeks with either placebo or bezafibrate in a double-blind cross-over design. Bezafibrate induced a significant decrease in plasma triglycerides (P less than 0.01), cholesterol (P less than 0.05), VLDL triglycerides (P less than 0.05) and VLDL cholesterol (P less than 0.01), and a significant increase in HDL cholesterol (P less than 0.01), whereas LDL cholesterol remained unchanged. The apolipoprotein AI/apolipoprotein B ratio increased significantly (P less than 0.05), although individual changes in these apolipoproteins were not significant. Apolipoprotein AII increased significantly (P less than 0.05), although individual changes in these apolipoproteins were not significant. Apolipoprotein AII increased significantly (P less than 0.0001) and the apolipoprotein AI/apolipoprotein AII ratio decreased (P less than 0.0001), indicating an increase in the HDL3 rather than the HDL2 fraction. No significant change in LCAT activity was observed.
Assuntos
Clofibrato/análogos & derivados , Ácido Clofíbrico/análogos & derivados , Diabetes Mellitus/tratamento farmacológico , Hiperlipoproteinemias/sangue , Fosfatidilcolina-Esterol O-Aciltransferase/sangue , Idoso , Apolipoproteína A-II , Apolipoproteínas/sangue , Apolipoproteínas A , Apolipoproteínas B , Bezafibrato , Colesterol/sangue , HDL-Colesterol , LDL-Colesterol , VLDL-Colesterol , Ácido Clofíbrico/uso terapêutico , Feminino , Humanos , Lipídeos/sangue , Lipoproteínas/sangue , Lipoproteínas HDL/sangue , Lipoproteínas LDL/sangue , Lipoproteínas VLDL/sangue , MasculinoRESUMO
Cardiovascular disease (CVD) in general seems to be the leading cause of death in the Eastern Mediterranean Region (EMR) including Iran. This may be due to classic risk factors such as high triglyceride (TG), high total cholesterol (TC), and low levels of high density lipoprotein cholesterol (HDL-C). The impact of antioxidants as potentially protective risk factors against early coronary heart disease (CHD) is unknown in Iran. Therefore, relationships between angina and plasma antioxidants and indicators of lipid peroxidation were investigated in a case-control study. In this study, 82 cases of previously undiagnosed angina pectoris (AP), identified by a modified WHO Rose chest pain questionnaire and verified by electrocardiography during treadmill exercise testing, were compared with 146 controls selected from the same population of over 4000 male civil servants aged 40-60 years. Subjects with AP declared significantly less physical activity and had higher serum TG [means (S.E.M.) 2.32 (0.18) versus 1.61 (0.07) mmol/l] but lower HDL-C [1.01 (0.04) versus 1.18 (0.03) mmol/l] than age-matched controls. Levels of total serum cholesterol, low-density lipoprotein cholesterol (LDL-C) and lipoprotein(a) [Lp(a)] were not significantly different between the two groups, while the ratio of LDL-C/HDL-C was significantly higher [4.51 (0.23) versus 3.54 (0. 11)] for subjects with AP than for the controls. There was no significant difference in plasma levels of alpha-tocopherol, vitamin C, alpha- and beta-carotene. However, retinol [1.90 (0.06) versus 2. 09 (0.05)] and beta-cryptoxanthin [0.398 (0.04) versus 0.467 (0.03)] were significantly lower in AP. Furthermore, angina cases exhibited a higher index of lipid peroxidation than controls (e.g. malondialdehyde, MDA; 0.376 (0.010) versus 0.337 (0.009) micromol/l). On multiple logistic regression analysis, retinol with odds ratio (OR) of 0.644 [95% confidence interval (CI; 0.425-0.978)], beta-cryptoxanthin, with an OR of 0.675 (CI; 0.487-0.940), oxidation indices, MDA with OR of 1.612 (95% CI; 1.119-2.322) and LDL-C/HDL-C ratio with OR of 2.006 (95% CI; 1.416-2.849) showed the most significant independent associations with AP in this group of Iranians. In conclusion, the state of lipid peroxidation as well as the status of special antioxidants may be co-determinants of AP in Iran, in parallel with the influence of classical risk factors for cardiovascular disease.