RESUMO
Recent evidence indicates ferroptosis is implicated in the pathophysiology of various liver diseases; however, the organ-specific regulation mechanism is poorly understood. Here, we demonstrate 7-dehydrocholesterol reductase (DHCR7), the terminal enzyme of cholesterol biosynthesis, as a regulator of ferroptosis in hepatocytes. Genetic and pharmacological inhibition (with AY9944) of DHCR7 suppress ferroptosis in human hepatocellular carcinoma Huh-7 cells. DHCR7 inhibition increases its substrate, 7-dehydrocholesterol (7-DHC). Furthermore, exogenous 7-DHC supplementation using hydroxypropyl ß-cyclodextrin suppresses ferroptosis. A 7-DHC-derived oxysterol metabolite, 3ß,5α-dihydroxycholest-7-en-6-one (DHCEO), is increased by the ferroptosis-inducer RSL-3 in DHCR7-deficient cells, suggesting that the ferroptosis-suppressive effect of DHCR7 inhibition is associated with the oxidation of 7-DHC. Electron spin resonance analysis reveals that 7-DHC functions as a radical trapping agent, thus protecting cells from ferroptosis. We further show that AY9944 inhibits hepatic ischemia-reperfusion injury, and genetic ablation of Dhcr7 prevents acetaminophen-induced acute liver failure in mice. These findings provide new insights into the regulatory mechanism of liver ferroptosis and suggest a potential therapeutic option for ferroptosis-related liver diseases.
Assuntos
Ferroptose , Hepatopatias , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Camundongos , Animais , Humanos , Dicloridrato de trans-1,4-Bis(2-clorobenzaminometil)ciclo-hexano , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismoRESUMO
BACKGROUND: 25-hydroxycholesterol (25HC), produced by cholesterol 25-hydroxylase (CH25H) in macrophages, has been reported to inhibit the replication of viral pathogens such as severe acute respiratory syndrome coronavirus-2. Also, CH25H expression in macrophages is robustly induced by interferons (IFNs). OBJECTIVE: To better understand the serum level increase of 25HC in coronavirus disease 2019 (COVID-19) and how it relates to the clinical picture. METHODS: We measured the serum levels of 25HC and five other oxysterols in 17 hospitalized COVID-19 patients. RESULTS: On admission, 25HC and 27-hydroxycholesterol (27HC) serum levels were elevated; however, 7-ketocholesterol (7KC) levels were lower in patients with COVID-19 than in the healthy controls. There was no significant correlation between 25HC serum levels and disease severity markers, such as interferon-gamma (IFN-γ) and interleukin 6. Dexamethasone effectively suppressed cholesterol 25-hydroxylase (CH25H) mRNA expression in RAW 264.7 cells, a murine leukemia macrophage cell line, with or without lipopolysaccharide or IFNs; therefore, it might mitigate the increasing effects of COVID-19 on the serum levels of 25HC. CONCLUSIONS: Our results highlighted that 25HC could be used as a unique biomarker in severe COVID-19 and a potential therapeutic candidate for detecting the severity of COVID-19 and other infectious diseases.
Assuntos
Antivirais , COVID-19 , Humanos , Animais , Camundongos , Antivirais/farmacologia , Replicação Viral , Linhagem CelularRESUMO
During obesity, tissue macrophages increase in number and become proinflammatory, thereby contributing to metabolic dysfunction. Lipoprotein lipase (LPL), which hydrolyzes triglyceride in lipoproteins, is secreted by macrophages. However, the role of macrophage-derived LPL in adipose tissue remodeling and lipoprotein metabolism is largely unknown. To clarify these issues, we crossed leptin-deficient Lepob/ob mice with mice lacking the Lpl gene in myeloid cells (Lplm-/m-) to generate Lplm-/m-;Lepob/ob mice. We found the weight of perigonadal white adipose tissue (WAT) was increased in Lplm-/m-;Lepob/ob mice compared with Lepob/ob mice due to substantial accumulation of both adipose tissue macrophages and collagen that surrounded necrotic adipocytes. In the fibrotic epidydimal WAT of Lplm-/m-;Lepob/ob mice, we observed an increase in collagen VI and high mobility group box 1, while α-smooth muscle cell actin, a marker of myofibroblasts, was almost undetectable, suggesting that the adipocytes were the major source of the collagens. Furthermore, the adipose tissue macrophages from Lplm-/m-;Lepob/ob mice showed increased expression of genes related to fibrosis and inflammation. In addition, we determined Lplm-/m-;Lepob/ob mice were more hypertriglyceridemic than Lepob/ob mice. Lplm-/m-;Lepob/ob mice also showed slower weight gain than Lepob/ob mice, which was primarily due to reduced food intake. In conclusion, we discovered that the loss of myeloid Lpl led to extensive fibrosis of perigonadal WAT and hypertriglyceridemia. In addition to illustrating an important role of macrophage LPL in regulation of circulating triglyceride levels, these data show that macrophage LPL protects against fibrosis in obese adipose tissues.
Assuntos
Tecido Adiposo Branco , Colágeno Tipo IV , Hipertrigliceridemia , Lipase Lipoproteica , Obesidade , Actinas/metabolismo , Tecido Adiposo Branco/patologia , Animais , Colágeno Tipo IV/metabolismo , Fibrose , Hipertrigliceridemia/genética , Hipertrigliceridemia/patologia , Leptina/deficiência , Leptina/genética , Lipase Lipoproteica/genética , Lipoproteínas/metabolismo , Camundongos , Camundongos Obesos , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologia , Triglicerídeos/sangueRESUMO
Valosin-containing protein (VCP) is a member of AAA-ATPase superfamily involved in various cellular functions. To investigate the pathophysiological role of VCP in metabolic disorders, we generated knock-in mice bearing an A232E mutation in VCP, a known human VCP pathogenic variant. When heterozygous mutant mice (A232E/+) were fed a high-fat diet, we observed that fatty liver was ameliorated and the proteolytic processing of the transcription factor sterol regulatory element-binding protein 1 (SREBP1) was impaired. Further co-immunoprecipitation analysis in wildtype mice revealed interactions of VCP with SREBP1 and a rhomboid protease, RHBDL4, in the liver, and these interactions were attenuated in A232E/+ mice. Consistent with these results, we show that knockdown or chemical inhibition of VCP or RHBDL4 in human hepatocytes impaired the proteolytic processing of SREBP1. Finally, we found that knockdown of E3 ligases such as glycoprotein 78 and HMG-CoA reductase degradation protein 1 disrupted the interaction of VCP with SREBP1 and impaired the proteolytic processing of SREBP1. These results suggest that VCP recognizes ubiquitinylated SREBP1 and recruits it to RHBDL4 to promote its proteolytic processing. The present study reveals a novel proteolytic processing pathway of SREBP1 and may lead to development of new therapeutic strategies to treat fatty liver diseases.
Assuntos
Proteínas de Membrana , Proteína de Ligação a Elemento Regulador de Esterol 1 , Proteína com Valosina , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Membrana/metabolismo , Camundongos , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína com Valosina/genética , Proteína com Valosina/metabolismoRESUMO
Leptin is an adipocyte-derived hormone that regulates appetite and energy expenditure via the hypothalamus. Since the majority of obese subjects are leptin resistant, leptin sensitizers, rather than leptin itself, are expected to be anti-obesity drugs. Endoplasmic reticulum (ER) stress in the hypothalamus plays a key role in the pathogenesis of leptin resistance. ATP-deficient cells are vulnerable to ER stress and ATP treatment protects cells against ER stress. Thus, we investigated the therapeutic effects of oral 1,3-butanediol (BD) administration, which increases plasma ß-hydroxybutyrate and hypothalamic ATP concentrations, in diet induced obese (DIO) mice with leptin resistance. BD treatment effectively decreased food intake and body weight in DIO mice. In contrast, BD treatment had no effect in leptin deficient ob/ob mice. Co-administration experiment demonstrated that BD treatment sensitizes leptin action in both DIO and ob/ob mice. We also demonstrated that BD treatment attenuates ER stress and leptin resistance at the hypothalamus level. This is the first report to confirm the leptin sensitizing effect of BD treatment in leptin resistant DIO mice. The present study provides collateral evidence suggesting that the effect of BD treatment is mediated by the elevation of hypothalamic ATP concentration. Ketone bodies and hypothalamic ATP are the potential target for the treatment of obesity and its complications.
Assuntos
Peso Corporal/efeitos dos fármacos , Butileno Glicóis/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Hipotálamo/efeitos dos fármacos , Leptina/farmacologia , Obesidade/tratamento farmacológico , Ácido 3-Hidroxibutírico/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Butileno Glicóis/uso terapêutico , Metabolismo Energético/efeitos dos fármacos , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Obesos , Obesidade/metabolismoRESUMO
Inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), statins, which are used to prevent cardiovascular diseases, are associated with a modest increase in the risk of new-onset diabetes. To investigate the role of HMGCR in the development of ß-cells and glucose homeostasis, we deleted Hmgcr in a ß-cell-specific manner by using the Cre-loxP technique. Mice lacking Hmgcr in ß-cells (ß-KO) exhibited hypoinsulinemic hyperglycemia as early as postnatal day 9 (P9) due to decreases in both ß-cell mass and insulin secretion. Ki67-positive cells were reduced in ß-KO mice at P9; thus, ß-cell mass reduction was caused by proliferation disorder immediately after birth. The mRNA expression of neurogenin3 (Ngn3), which is transiently expressed in endocrine progenitors of the embryonic pancreas, was maintained despite a striking reduction in the expression of ß-cell-associated genes, such as insulin, pancreatic and duodenal homeobox 1 (Pdx1), and MAF BZIP transcription factor A (Mafa) in the islets from ß-KO mice. Histological analyses revealed dysmorphic islets with markedly reduced numbers of ß-cells, some of which were also positive for glucagon. In conclusion, HMGCR plays critical roles not only in insulin secretion but also in the development of ß-cells in mice.
Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Hidroximetilglutaril-CoA Redutases/metabolismo , Células Secretoras de Insulina/enzimologia , Insulina/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Glicemia , Diabetes Mellitus , Comportamento Alimentar , Teste de Tolerância a Glucose , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hidroximetilglutaril-CoA Redutases/genética , Hiperglicemia , Insulina/sangue , Células Secretoras de Insulina/metabolismo , Fatores de Transcrição Maf Maior/genética , Fatores de Transcrição Maf Maior/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
Disturbance of circadian rhythms underlies various metabolic diseases. Constant light exposure (LL) is known to disrupt both central and peripheral circadian rhythms. Here, we attempted to determine whether the effects of LL are different between various peripheral tissues and whether time-restricted feeding restores the circadian rhythms especially in white adipose tissue (WAT). Six-week-old mice were subjected to three feeding regimes: ad libitum feeding under light/dark phase (LD), ad libitum feeding under LL cycle, and restricted feeding at night-time under LL cycle with a normal chow. After 3 weeks, we compared body weight, food intake, plasma levels of lipids and glucose, and the expression patterns of the clock genes and the genes involved in lipid metabolism in the liver and WAT. The mice kept under LL with or without time-restricted feeding were 5.2% heavier (p<0.001, n = 16) than the mice kept under LD even though the food intakes of the two groups were the same. Food intake occurred mostly in the dark phase. LL disrupted this pattern, causing disruptions in circadian rhythms of plasma levels of triglycerides (TG) and glucose. Time-restricted feeding partially restored the rhythms. LL eliminated the circadian rhythms of the expression of the clock genes as well as most of the genes involved in lipid metabolism in both liver and WAT. More notably, LL markedly decreased not only the amplitude but also the average levels of the expression of the genes in the liver, but not in the WAT, suggesting that transcription in the liver is sensitive to constant light exposure. Time-restricted feeding restored the circadian rhythms of most of the genes to various degrees in both liver and WAT. In conclusion, LL disrupted the peripheral circadian rhythms more severely in liver than in WAT. Time-restricted feeding restored the circadian rhythms in both tissues.
Assuntos
Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/genética , Ritmo Circadiano/fisiologia , Jejum/fisiologia , Luz/efeitos adversos , Metabolismo dos Lipídeos/fisiologia , Tecido Adiposo Branco/metabolismo , Animais , Ritmo Circadiano/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Metabolismo dos Lipídeos/efeitos da radiação , Fígado/metabolismo , Masculino , Camundongos , Modelos Animais , FotoperíodoRESUMO
The acyltransferase LCAT mediates FA esterification of plasma cholesterol. In vitro studies have shown that LCAT also FA-esterifies several oxysterols, but in vivo evidence is lacking. Here, we measured both free and FA-esterified forms of sterols in 206 healthy volunteers and 8 individuals with genetic LCAT deficiency, including familial LCAT deficiency (FLD) and fish-eye disease (FED). In the healthy volunteers, the mean values of the ester-to-total molar ratios of the following sterols varied: 4ß-hydroxycholesterol (4ßHC), 0.38; 5,6α-epoxycholesterol (5,6αEC), 0.46; 5,6ß-epoxycholesterol (5,6ßEC), 0.51; cholesterol, 0.70; cholestane-3ß,5α,6ß-triol (CT), 0.70; 7-ketocholesterol (7KC), 0.75; 24S-hydroxycholesterol (24SHC), 0.80; 25-hydroxycholesterol (25HC), 0.81; 27-hydroxycholesterol (27HC), 0.86; and 7α-hydroxycholesterol (7αHC), 0.89. In the individuals with LCAT deficiency, the plasma levels of the FA-esterified forms of cholesterol, 5,6αEC, 5,6ßEC, CT, 7αHC, 7KC, 24SHC, 25HC, and 27HC, were significantly lower than those in the healthy volunteers. The individuals with FLD had significantly lower FA-esterified forms of 7αHC, 24SHC, and 27HC than those with FED. It is of note that, even in the three FLD individuals with negligible plasma cholesteryl ester, substantial amounts of the FA-esterified forms of 4ßHC, 5,6αEC, 7αHC, 7KC, and 27HC were present. We conclude that LCAT has a major role in the FA esterification of many plasma oxysterols but contributes little to the FA esterification of 4ßHC. Substantial FA esterification of 4ßHC, 5,6αEC, 7αHC, 7KC, and 27HC is independent of LCAT.
Assuntos
Hidroxicolesteróis/sangue , Hidroxicolesteróis/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Adulto , Estudos de Casos e Controles , Esterificação , Feminino , Humanos , Masculino , Fosfatidilcolina-Esterol O-Aciltransferase/genética , Adulto JovemRESUMO
Adipose tissue macrophages (ATMs) are involved in the development of insulin resistance in obesity. We have recently shown that myeloid cell-specific reduction of HMG-CoA reductase (Hmgcr m-/m- ), which is the rate-limiting enzyme in cholesterol biosynthesis, protects against atherosclerosis by inhibiting macrophage migration in mice. We hypothesized that ATMs are harder to accumulate in Hmgcr m-/m- mice than in control Hmgcr fl/fl mice in the setting of obesity. To test this hypothesis, we fed Hmgcr m-/m- and Hmgcr fl/fl mice a high-fat diet (HFD) for 24 weeks and compared plasma glucose metabolism as well as insulin signaling and histology between the two groups. Myeloid cell-specific reduction of Hmgcr improved glucose tolerance and insulin sensitivity without altering body weight in the HFD-induced obese mice. The improvement was due to a decrease in the number of ATMs. The ATMs were reduced by decreased recruitment of macrophages as a result of their impaired chemotactic activity. These changes were associated with decreased expression of proinflammatory cytokines in adipose tissues. Myeloid cell-specific reduction of Hmgcr also attenuated hepatic steatosis. In conclusion, reducing myeloid HMGCR may be a promising strategy to improve insulin resistance and hepatic steatosis in obesity.
Assuntos
Tecido Adiposo/efeitos dos fármacos , Hidroximetilglutaril-CoA Redutases/metabolismo , Inflamação/metabolismo , Resistência à Insulina , Células Mieloides/metabolismo , Obesidade/induzido quimicamente , Tecido Adiposo/patologia , Animais , Glicemia , Dieta Hiperlipídica/efeitos adversos , Fígado Gorduroso/induzido quimicamente , Regulação Enzimológica da Expressão Gênica , Hidroximetilglutaril-CoA Redutases/genética , Inflamação/induzido quimicamente , Insulina/sangue , Macrófagos , Camundongos , Camundongos KnockoutRESUMO
AIM: Acyl-CoA cholesterol acyltransferase 1 (ACAT1) esterifies free cholesterol to cholesteryl esters (CE), which are subsequently hydrolyzed by neutral cholesterol ester hydrolase 1 (NCEH1). The elimination of ACAT1 in vitro reduces the amounts of CE accumulated in Nceh1-deficient macrophages. The present study aimed at examining whether the loss of ACAT1 attenuates atherosclerosis which is aggravated by the loss of NCEH1 in vivo. METHODS: Low density lipoprotein receptor (Ldlr)-deficient mice were transplanted with bone marrow from wild-type mice and mice lacking ACAT1, NCEH1, or both. The four types of mice were fed a high-cholesterol diet and, then, were examined for atherosclerosis. RESULTS: The cross-sectional lesion size of the recipients of Nceh1-deficient bone marrow was 1.6-fold larger than that of the wild-type bone marrow. The lesions of the recipients of Nceh1-deficient bone marrow were enriched with MOMA2-positive macrophages compared with the lesions of the recipients of the wild-type bone marrow. The size and the macrophage content of the lesions of the recipients of bone marrow lacking both ACAT1 and NCEH1 were significantly smaller than the recipients of the Nceh1-deficient bone marrow, indicating that the loss of ACAT1 decreases the excess CE in the Nceh1-deficient lesions. The collagen-rich and/or mucin-rich areas and en face lesion size were enlarged in the recipients of the Acat1ï¼/ï¼ bone marrow compared with those of the recipients of the WT bone marrow. CONCLUSION: The loss of ACAT1 in bone marrow-derived cells attenuates atherosclerosis, which is aggravated by the loss of NCEH1, corroborating the in vitro functions of ACAT1 (formation of CE) and NCEH1 (hydrolysis of CE).
Assuntos
Aterosclerose/etiologia , Transplante de Medula Óssea/efeitos adversos , Macrófagos Peritoneais/patologia , Receptores de LDL/fisiologia , Esterol Esterase/fisiologia , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Células Cultivadas , Colesterol/metabolismo , Estudos Transversais , Feminino , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Esterol O-AciltransferaseRESUMO
Objective- ACAT1 (Acyl-CoA cholesterol acyltransferase 1) esterifies cellular free cholesterol, thereby converting macrophages to cholesteryl ester-laden foam cells in atherosclerotic lesions and cutaneous xanthoma. Paradoxically, however, loss of ACAT1 in bone marrow causes the aggravation of atherosclerosis and the development of severe cutaneous xanthoma in hyperlipidemic mice. Recently, it has been reported that cholesterol crystals activate NLRP3 (NACHT, LRR [leucine-rich repeats], and PYD [pyrin domain] domain-containing protein 3) inflammasomes, thereby contributing to the development of atherosclerosis. The present study aimed to clarify the role of NLRP3 inflammasomes in the worsening of atherosclerosis and cutaneous xanthoma induced by ACAT1 deficiency. Approach and Results- Ldlr-null mice were transplanted with bone marrow from WT (wild type) mice and mice lacking ACAT1, NLRP3, or both. After the 4 types of mice were fed high-cholesterol diets, we compared their atherosclerosis and skin lesions. The mice transplanted with Acat1-null bone marrow developed severe cutaneous xanthoma, which was filled with numerous macrophages and cholesterol clefts and had markedly increased expression of inflammatory cytokines, and increased atherosclerosis. Loss of NLRP3 completely reversed the cutaneous xanthoma, whereas it improved the atherosclerosis only partially. Acat1-null peritoneal macrophages showed enhanced expression of CHOP (C/EBP [CCAAT/enhancer binding protein] homologous protein) and TNF-α (tumor necrosis factor-α) but no evidence of inflammasome activation, after treatment with acetylated LDL (low-density lipoprotein). Conclusions- Elimination of ACAT1 in bone marrow-derived cells aggravates cutaneous xanthoma and atherosclerosis. The development of cutaneous xanthoma is induced mainly via the NLRP3 inflammasome activation.
Assuntos
Acetil-CoA C-Acetiltransferase/metabolismo , Doenças da Aorta/enzimologia , Aterosclerose/enzimologia , Medula Óssea/enzimologia , Inflamassomos/metabolismo , Macrófagos Peritoneais/enzimologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Placa Aterosclerótica , Dermatopatias/enzimologia , Xantomatose/enzimologia , Acetil-CoA C-Acetiltransferase/deficiência , Acetil-CoA C-Acetiltransferase/genética , Animais , Doenças da Aorta/genética , Doenças da Aorta/patologia , Doenças da Aorta/prevenção & controle , Aterosclerose/genética , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Medula Óssea/patologia , Transplante de Medula Óssea , Células Cultivadas , Colesterol na Dieta , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Macrófagos Peritoneais/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/deficiência , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Fenótipo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Transdução de Sinais , Dermatopatias/genética , Dermatopatias/patologia , Dermatopatias/prevenção & controle , Xantomatose/genética , Xantomatose/patologia , Xantomatose/prevenção & controleRESUMO
Objective- Inhibition of HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) is atheroprotective primarily by decreasing plasma LDL (low-density lipoprotein)-cholesterol. However, it is unknown whether inhibition of HMGCR in myeloid cells contributes to this atheroprotection. We sought to determine the role of myeloid HMGCR in the development of atherosclerosis. Approach and Results- We generated mice with genetically reduced Hmgcr in myeloid cells ( Hmgcr m-/m-) using LysM (Cre) and compared various functions of their macrophages to those of Hmgcr fl/fl control mice. We further compared the extent of atherosclerosis in Hmgcr m-/ m- and Hmgcr fl/fl mice in the absence of Ldlr (LDL receptor). Hmgcr m-/ m- macrophages and granulocytes had significantly lower Hmgcr mRNA expression and cholesterol biosynthesis than Hmgcr fl/fl cells. In vitro, Hmgcr m-/ m- monocytes/macrophages had reduced ability to migrate, proliferate, and survive compared with Hmgcr fl/fl monocytes/macrophages. However, there was no difference in ability to adhere, phagocytose, store lipids, or polarize to M1 macrophages between the 2 types of macrophages. The amounts of plasma membrane-associated small GTPase proteins, such as RhoA (RAS homolog family member A), were increased in Hmgcr m-/ m- macrophages. In the setting of Ldlr deficiency, Hmgcr m-/ m- mice developed significantly smaller atherosclerotic lesions than Hmgcr fl/fl mice. However, there were no differences between the 2 types of mice either in plasma lipoprotein profiles or in the numbers of proliferating or apoptotic cells in the lesions in vivo. The in vivo migration of Hmgcr m-/ m- macrophages to the lesions was reduced compared with Hmgcr fl/fl macrophages. Conclusions- Genetic reduction of HMGCR in myeloid cells may exert atheroprotective effects primarily by decreasing the migratory activity of monocytes/macrophages to the lesions.
Assuntos
Aorta/enzimologia , Doenças da Aorta/enzimologia , Aterosclerose/enzimologia , Movimento Celular , Hidroximetilglutaril-CoA Redutases/metabolismo , Macrófagos Peritoneais/enzimologia , Monócitos/enzimologia , Transferência Adotiva , Animais , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Doenças da Aorta/prevenção & controle , Aterosclerose/genética , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Hidroximetilglutaril-CoA Redutases/genética , Lipídeos/sangue , Macrófagos Peritoneais/patologia , Macrófagos Peritoneais/transplante , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/patologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fenótipo , Receptores de LDL/deficiência , Receptores de LDL/genética , Transdução de SinaisRESUMO
Squalene synthase (SS) catalyzes the biosynthesis of squalene, the first specific intermediate in the cholesterol biosynthetic pathway. To test the feasibility of lowering plasma cholesterol by inhibiting hepatic SS, we generated mice in which SS is specifically knocked out in the liver (L-SSKO) using Cre-loxP technology. Hepatic SS activity of L-SSKO mice was reduced by >90%. In addition, cholesterol biosynthesis in the liver slices was almost eliminated. Although the hepatic squalene contents were markedly reduced in L-SSKO mice, the hepatic contents of cholesterol and its precursors distal to squalene were indistinguishable from those of control mice, indicating the presence of sufficient centripetal flow of cholesterol and/or its precursors from the extrahepatic tissues. L-SSKO mice showed a transient liver dysfunction with moderate hepatomegaly presumably secondary to increased farnesol production. In a fed state, the plasma total cholesterol and triglyceride were significantly reduced in L-SSKO mice, primarily owing to reduced hepatic VLDL secretion. In a fasted state, the hypolipidemic effect was lost. mRNA expression of liver X receptor α target genes was reduced, while that of sterol-regulatory element binding protein 2 target genes was increased. In conclusion, liver-specific ablation of SS inhibits hepatic cholesterol biosynthesis and induces hypolipidemia without increasing significant mortality.
Assuntos
Colesterol/sangue , Farnesil-Difosfato Farnesiltransferase/genética , Fígado/enzimologia , Animais , Vias Biossintéticas , Colesterol/biossíntese , Farnesil-Difosfato Farnesiltransferase/metabolismo , Hidroximetilglutaril-CoA Redutases/metabolismo , Fígado/fisiopatologia , Masculino , Camundongos TransgênicosRESUMO
5-(4'-Methoxyphenyl)-oxazole (MPO), originally reported as a synthetic compound, was isolated from fungal culture broth as an inhibitor of hatch and growth of Caenorhabditis elegans. Nineteen MPO derivatives were chemically synthesized, but showed no effect on C. elegans hatch and growth. These findings strongly suggested that the whole structure of MPO is essential for anti-C. elegans activity.
Assuntos
Antinematódeos/síntese química , Antinematódeos/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/crescimento & desenvolvimento , Oxazóis/química , Animais , Bactérias/efeitos dos fármacos , Células Cultivadas , Fungos/efeitos dos fármacos , Células Germinativas/citologia , Células Germinativas/efeitos dos fármacos , Células HeLa , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
An excess of cholesterol and/or oxysterols induces apoptosis in macrophages, contributing to the development of advanced atherosclerotic lesions. In foam cells, these sterols are stored in esterified forms, which are hydrolyzed by two enzymes: neutral cholesterol ester hydrolase 1 (Nceh1) and hormone-sensitive lipase (Lipe). A deficiency in either enzyme leads to accelerated growth of atherosclerotic lesions in mice. However, it is poorly understood how the esterification and hydrolysis of sterols are linked to apoptosis. Remarkably, Nceh1-deficient thioglycollate-elicited peritoneal macrophages (TGEMs), but not Lipe-deficient TGEMs, were more susceptible to apoptosis induced by oxysterols, particularly 25-hydroxycholesterol (25-HC), and incubation with 25-HC caused massive accumulation of 25-HC ester in the endoplasmic reticulum (ER) due to its defective hydrolysis, thereby activating ER stress signaling such as induction of CCAAT/enhancer-binding protein-homologous protein (CHOP). These changes were nearly reversed by inhibition of ACAT1. In conclusion, deficiency of Nceh1 augments 25-HC-induced ER stress and subsequent apoptosis in TGEMs. In addition to reducing the cholesteryl ester content of foam cells, Nceh1 may protect against the pro-apoptotic effect of oxysterols and modulate the development of atherosclerosis.
Assuntos
Apoptose , Estresse do Retículo Endoplasmático , Hidroxicolesteróis/metabolismo , Macrófagos Peritoneais/enzimologia , Transdução de Sinais , Esterol Esterase/metabolismo , Acetil-CoA C-Acetiltransferase/genética , Acetil-CoA C-Acetiltransferase/metabolismo , Animais , Aterosclerose/enzimologia , Aterosclerose/genética , Aterosclerose/patologia , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/patologia , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Knockout , Esterol Esterase/genética , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismoRESUMO
Hydrolysis of intracellular cholesteryl ester (CE) is the rate-limiting step in the efflux of cholesterol from macrophage foam cells. In mouse peritoneal macrophages (MPMs), this process is thought to involve several enzymes: hormone-sensitive lipase (Lipe), carboxylesterase 3 (Ces3), neutral CE hydrolase 1 (Nceh1). However, there is some disagreement over the relative contributions of these enzymes. To solve this problem, we first compared the abilities of several compounds to inhibit the hydrolysis of CE in cells overexpressing Lipe, Ces3, or Nceh1. Cells overexpressing Ces3 had negligible neutral CE hydrolase activity. We next examined the effects of these inhibitors on the hydrolysis of CE and subsequent cholesterol trafficking in MPMs. CE accumulation was increased by a selective inhibitor of Nceh1, paraoxon, and two nonselective inhibitors of Nceh1, (+)-AS115 and (-)-AS115, but not by two Lipe-selective inhibitors, orlistat and 76-0079. Paraoxon inhibited cholesterol efflux to apoA-I or HDL, while 76-0079 did not. These results suggest that Nceh1 plays a dominant role over Lipe in the hydrolysis of CE and subsequent cholesterol efflux in MPMs.
Assuntos
Ésteres do Colesterol/metabolismo , Macrófagos Peritoneais/enzimologia , Esterol Esterase/metabolismo , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/genética , Carboxilesterase/genética , Carboxilesterase/metabolismo , Ésteres do Colesterol/genética , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Hidrólise , Camundongos , Camundongos Knockout , Esterol Esterase/antagonistas & inibidores , Esterol Esterase/genéticaRESUMO
Microbial samples, including our library of known microbial compounds (ca. 300) and microbial culture broths (ca. 9000), were screened for small molecules affecting the phenotype of Caenorhabditis elegans. As a result, seven known compounds were found to induce phenotypic abnormality of C. elegans. Staurosporine exhibited morphological defects in the vulva and tail of C. elegans, avermectin B1a exhibited hatching inhibition of starting eggs on day 1 at 25-100 µM and growth inhibition at 0.01-12.5 µM, siccanin and antimycin A inhibited the growth of C. elegans, and fluorouracil inhibited hatching of eggs newly spawned by adult C. elegans. Toromycin induced morphological defects in the intestine. 5-(4-Methoxyphenyl)-oxazole, isolated as a fungal metabolite for the first time, inhibited the hatching of eggs newly spawned by adult C. elegans.