RESUMO
It is known that high-fat diet (HFD) and/or diabetes may influence substrate preferences and energy demands in the heart preceding diabetic cardiomyopathy. They may also induce structural glomerular changes causing diabetic nephropathy. PET/CT has been utilized to examine uptake of energy substrates, and to study metabolic changes or shifts before onset of metabolic disorders. However, conventional PET/CT scanning of organs with relatively low uptake, such as the kidney, in small animals in vivo may render technical difficulties. To address this issue, we developed a PET/CT ex vivo protocol with radiolabeled glucose and fatty acid analouges, [18F]FDG and [18F]FTHA,to study substrate uptake in mouse kidneys. We also aimed to detect a possible energy substrate shift before onset of diabetic nephropathy. The ex vivo protocol reduced interfering background as well as interindividual variances. We found increased uptake of [18F]FDG and [18F]FTHA in kidneys after HFD, compared to kidneys from young mice on standard chow. Levels of kidney triglycerides also increased on HFD. Lipoprotein lipase (LPL) activity, the enzyme responsible for release of fatty acids from circulating lipoproteins, is normally increased in postprandial mice kidneys. After long-term HFD, we found that LPL activity was suppressed, and could therefore not explain the increased levels of stored triglycerides. Suppressed LPL activity was associated with increased expression of angiopoietin-like protein4, an inhibitor of LPL. HFD did not alter the transcriptional control of some common glucose and fatty acid transporters that may mediate uptake of [18F]FDG and [18F]FTHA. Performing PET/CT ex vivo reduced interfering background and interindividual variances. Obesity and insulin resistance induced by HFD increased the uptake of [18F]FDG and [18F]FTHA and triglyceride accumulation in mouse kidneys. Increased levels of [18F]FDG and [18F]FTHA in obese insulin resistant mice could be used clinically as an indicator of poor metabolic control, and a complementary test for incipient diabetic nephropathy.
Assuntos
Nefropatias Diabéticas , Fluordesoxiglucose F18 , Animais , Camundongos , Dieta Hiperlipídica , Ácidos Graxos/metabolismo , Glucose/metabolismo , Rim/diagnóstico por imagem , Rim/metabolismo , Camundongos Endogâmicos C57BL , Obesidade/diagnóstico por imagem , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , TriglicerídeosRESUMO
BACKGROUND: Activity assays for lipoprotein lipase (LPL) are not standardised for use in clinical settings. OBJECTIVE: This study sought to define and validate a cut-off points based on a ROC curve for the diagnosis of patients with familial chylomicronemia syndrome (FCS). We also evaluated the role of LPL activity in a comprehensive FCS diagnostic workflow. METHODS: A derivation cohort (including an FCS group (n = 9), a multifactorial chylomicronemia syndrome (MCS) group (n = 11)), and an external validation cohort (including an FCS group (n = 5), a MCS group (n = 23) and a normo-triglyceridemic (NTG) group (n = 14)), were studied. FCS patients were previously diagnosed by the presence of biallelic pathogenic genetic variants in the LPL and GPIHBP1 genes. LPL activity was also measured. Clinical and anthropometric data were recorded, and serum lipids and lipoproteins were measured. Sensitivity, specificity and cut-offs for LPL activity were obtained from a ROC curve and externally validated. RESULTS: All post-heparin plasma LPL activity in the FCS patients were below 25.1 mU/mL, that was cut-off with best performance. There was no overlap in the LPL activity distributions between the FCS and MCS groups, conversely to the FCS and NTG groups. CONCLUSION: We conclude that, in addition to genetic testing, LPL activity in subjects with severe hypertriglyceridemia is a reliable criterium in the diagnosis of FCS when using a cut-off of 25.1 mU/mL (25% of the mean LPL activity in the validation MCS group). We do not recommend the NTG patient based cut-off values due to low sensitivity.
Assuntos
Hiperlipoproteinemia Tipo I , Hipertrigliceridemia , Receptores de Lipoproteínas , Humanos , Hiperlipoproteinemia Tipo I/diagnóstico , Hiperlipoproteinemia Tipo I/genética , Lipase Lipoproteica/genética , Hipertrigliceridemia/genética , Testes Genéticos , Receptores de Lipoproteínas/genética , TriglicerídeosRESUMO
Angiopoietin-like 4 (ANGPTL4) is an important regulator of plasma triglyceride (TG) levels and an attractive pharmacological target for lowering plasma lipids and reducing cardiovascular risk. Here, we aimed to study the efficacy and safety of silencing ANGPTL4 in the livers of mice using hepatocyte-targeting GalNAc-conjugated antisense oligonucleotides (ASOs). Compared with injections with negative control ASO, four injections of two different doses of ANGPTL4 ASO over 2 weeks markedly downregulated ANGPTL4 levels in liver and adipose tissue, which was associated with significantly higher adipose LPL activity and lower plasma TGs in fed and fasted mice, as well as lower plasma glucose levels in fed mice. In separate experiments, injection of two different doses of ANGPTL4 ASO over 20 weeks of high-fat feeding reduced hepatic and adipose ANGPTL4 levels but did not trigger mesenteric lymphadenopathy, an acute phase response, chylous ascites, or any other pathological phenotypes. Compared with mice injected with negative control ASO, mice injected with ANGPTL4 ASO showed reduced food intake, reduced weight gain, and improved glucose tolerance. In addition, they exhibited lower plasma TGs, total cholesterol, LDL-C, glucose, serum amyloid A, and liver TG levels. By contrast, no significant difference in plasma alanine aminotransferase activity was observed. Overall, these data suggest that ASOs targeting ANGPTL4 effectively reduce plasma TG levels in mice without raising major safety concerns.
Assuntos
Glucose , Linfadenopatia , Proteína 4 Semelhante a Angiopoietina/genética , Animais , Camundongos , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , TriglicerídeosRESUMO
LPL is a key player in plasma triglyceride metabolism. Consequently, LPL is regulated by several proteins during synthesis, folding, secretion, and transport to its site of action at the luminal side of capillaries, as well as during the catalytic reaction. Some proteins are well known, whereas others have been identified but are still not fully understood. We set out to study the effects of the natural variations in the plasma levels of all known LPL regulators on the activity of purified LPL added to samples of fasted plasma taken from 117 individuals. The enzymatic activity was measured at 25°C using isothermal titration calorimetry. This method allows quantification of the ability of an added fixed amount of exogenous LPL to hydrolyze triglyceride-rich lipoproteins in plasma samples by measuring the heat produced. Our results indicate that, under the conditions used, the normal variation in the endogenous levels of apolipoprotein C1, C2, and C3 or the levels of angiopoietin-like proteins 3, 4, and 8 in the fasted plasma samples had no significant effect on the recorded activity of the added LPL. Instead, the key determinant for the LPL activity was a lipid signature strongly correlated to the average size of the VLDL particles. The signature involved not only several lipoprotein and plasma lipid parameters but also apolipoprotein A5 levels. While the measurements cannot fully represent the action of LPL when attached to the capillary wall, our study provides knowledge on the interindividual variation of LPL lipolysis rates in human plasma.
Assuntos
Lipoproteínas , TriglicerídeosRESUMO
OBJECTIVE: Studies in mice have shown that the decrease in lipoprotein lipase (LPL) activity in adipose tissue upon fasting is mediated by induction of the inhibitor ANGPTL4. Here, we aimed to validate this concept in humans by determining the effect of a prolonged fast on ANGPTL4 and LPL gene and protein expression in human subcutaneous adipose tissue. METHODS: Twenty-three volunteers ate a standardized meal at 18.00 h and fasted until 20.00 h the next day. Blood was drawn and periumbilical adipose tissue biopsies were collected 2 h and 26 h after the meal. RESULTS: Consistent with previous mouse data, LPL activity in human adipose tissue was significantly decreased by fasting (-60%), concurrent with increased ANGPTL4 mRNA (+90%) and decreased ANGPTL8 mRNA (-94%). ANGPTL4 protein levels in adipose tissue were also significantly increased by fasting (+46%), whereas LPL mRNA and protein levels remained unchanged. In agreement with the adipose tissue data, plasma ANGPTL4 levels increased upon fasting (+100%), whereas plasma ANGPTL8 decreased (-79%). Insulin, levels of which significantly decreased upon fasting, downregulated ANGPTL4 mRNA and protein in primary human adipocytes. By contrast, cortisol, levels of which significantly increased upon fasting, upregulated ANGPTL4 mRNA and protein in primary human adipocytes as did fatty acids. CONCLUSION: ANGPTL4 levels in human adipose tissue are increased by fasting, likely via increased plasma cortisol and free fatty acids and decreased plasma insulin, resulting in decreased LPL activity. This clinical trial was registered with identifier NCT03757767.
Assuntos
Proteína 4 Semelhante a Angiopoietina/metabolismo , Jejum/metabolismo , Lipase Lipoproteica/metabolismo , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Adulto , Idoso , Proteína 4 Semelhante a Angiopoietina/fisiologia , Proteína 8 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina/metabolismo , Ácidos Graxos/metabolismo , Ácidos Graxos não Esterificados/análise , Ácidos Graxos não Esterificados/sangue , Feminino , Humanos , Insulina/metabolismo , Resistência à Insulina/fisiologia , Lipase Lipoproteica/fisiologia , Masculino , Pessoa de Meia-Idade , Hormônios Peptídicos/metabolismo , Triglicerídeos/análise , Triglicerídeos/metabolismoRESUMO
The binding of lipoprotein lipase (LPL) to GPIHBP1 focuses the intravascular hydrolysis of triglyceride-rich lipoproteins on the surface of capillary endothelial cells. This process provides essential lipid nutrients for vital tissues (e.g., heart, skeletal muscle, and adipose tissue). Deficiencies in either LPL or GPIHBP1 impair triglyceride hydrolysis, resulting in severe hypertriglyceridemia. The activity of LPL in tissues is regulated by angiopoietin-like proteins 3, 4, and 8 (ANGPTL). Dogma has held that these ANGPTLs inactivate LPL by converting LPL homodimers into monomers, rendering them highly susceptible to spontaneous unfolding and loss of enzymatic activity. Here, we show that binding of an LPL-specific monoclonal antibody (5D2) to the tryptophan-rich lipid-binding loop in the carboxyl terminus of LPL prevents homodimer formation and forces LPL into a monomeric state. Of note, 5D2-bound LPL monomers are as stable as LPL homodimers (i.e., they are not more prone to unfolding), but they remain highly susceptible to ANGPTL4-catalyzed unfolding and inactivation. Binding of GPIHBP1 to LPL alone or to 5D2-bound LPL counteracts ANGPTL4-mediated unfolding of LPL. In conclusion, ANGPTL4-mediated inactivation of LPL, accomplished by catalyzing the unfolding of LPL, does not require the conversion of LPL homodimers into monomers. Thus, our findings necessitate changes to long-standing dogma on mechanisms for LPL inactivation by ANGPTL proteins. At the same time, our findings align well with insights into LPL function from the recent crystal structure of the LPLâ¢GPIHBP1 complex.
Assuntos
Proteína 4 Semelhante a Angiopoietina/metabolismo , Lipase Lipoproteica/química , Triglicerídeos/sangue , Motivos de Aminoácidos , Proteína 4 Semelhante a Angiopoietina/genética , Animais , Anticorpos Monoclonais/metabolismo , Dimerização , Humanos , Hipertrigliceridemia/enzimologia , Hipertrigliceridemia/genética , Hipertrigliceridemia/metabolismo , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Desdobramento de Proteína , Receptores de Lipoproteínas/química , Receptores de Lipoproteínas/genética , Receptores de Lipoproteínas/metabolismoRESUMO
CONTEXT: Maternal body mass index (BMI) is associated with increased birth weight but does not explain all the variance in fetal adiposity. OBJECTIVE: To assess the contribution of maternal body fat distribution to offspring birth weight and adiposity. DESIGN: Longitudinal study throughout gestation and at delivery. SETTING: Women recruited at 12 weeks of gestation and followed up at 26 and 36 weeks. Cord blood was collected at delivery. PATIENTS: Pregnant women (n = 45) with BMI 18.0 to 46.3 kg/m2 and healthy pregnancy outcome. METHODS: Maternal first trimester abdominal subcutaneous and visceral adipose tissue thickness (SAT and VAT) was assessed by ultrasound. MAIN OUTCOME MEASURES: Maternal body fat distribution, maternal and cord plasma glucose and lipid concentrations, placental weight, birth weight, and fetal adiposity assessed by cord blood leptin. RESULTS: VAT was the only anthropometric measure independently associated with birth weight centile (r2 adjusted 15.8%, P = .002). BMI was associated with trimester 2 and trimesters 1 through 3 area under the curve (AUC) glucose and insulin resistance (Homeostatic Model Assessment). SAT alone predicted trimester 2 lipoprotein lipase (LPL) mass (a marker of adipocyte insulin sensitivity) (11.3%, P = .017). VAT was associated with fetal triglyceride (9.3%, P = .047). Placental weight was the only independent predictor of fetal adiposity (48%, P < .001). Maternal trimester 2 and AUC LPL were inversely associated with fetal adiposity (r = -0.69, P = .001 and r = -0.58, P = .006, respectively). CONCLUSIONS: Maternal VAT provides additional information to BMI for prediction of birth weight. VAT may be a marker of reduced SAT expansion and increased availability of maternal fatty acids for placental transport.
Assuntos
Adiposidade/fisiologia , Peso ao Nascer/fisiologia , Feto/fisiologia , Gordura Intra-Abdominal/fisiologia , Trimestres da Gravidez/fisiologia , Adulto , Área Sob a Curva , Glicemia/metabolismo , Distribuição da Gordura Corporal , Índice de Massa Corporal , Feminino , Humanos , Recém-Nascido , Resistência à Insulina , Gordura Intra-Abdominal/diagnóstico por imagem , Estudos Longitudinais , Gravidez , Ultrassonografia Pré-NatalRESUMO
Angiopoietin-like protein (ANGPTL)4 regulates plasma lipids, making it an attractive target for correcting dyslipidemia. However, ANGPTL4 inactivation in mice fed a high fat diet causes chylous ascites, an acute-phase response, and mesenteric lymphadenopathy. Here, we studied the role of ANGPTL4 in lipid uptake in macrophages and in the above-mentioned pathologies using Angptl4-hypomorphic and Angptl4-/- mice. Angptl4 expression in peritoneal and bone marrow-derived macrophages was highly induced by lipids. Recombinant ANGPTL4 decreased lipid uptake in macrophages, whereas deficiency of ANGPTL4 increased lipid uptake, upregulated lipid-induced genes, and increased respiration. ANGPTL4 deficiency did not alter LPL protein levels in macrophages. Angptl4-hypomorphic mice with partial expression of a truncated N-terminal ANGPTL4 exhibited reduced fasting plasma triglyceride, cholesterol, and NEFAs, strongly resembling Angptl4-/- mice. However, during high fat feeding, Angptl4-hypomorphic mice showed markedly delayed and attenuated elevation in plasma serum amyloid A and much milder chylous ascites than Angptl4-/- mice, despite similar abundance of lipid-laden giant cells in mesenteric lymph nodes. In conclusion, ANGPTL4 deficiency increases lipid uptake and respiration in macrophages without affecting LPL protein levels. Compared with the absence of ANGPTL4, low levels of N-terminal ANGPTL4 mitigate the development of chylous ascites and an acute-phase response in mice.
Assuntos
Adipócitos/metabolismo , Proteína 4 Semelhante a Angiopoietina/deficiência , Proteína 4 Semelhante a Angiopoietina/genética , Técnicas de Inativação de Genes , Macrófagos/metabolismo , Animais , Respiração Celular , Ascite Quilosa/genética , Ascite Quilosa/patologia , Éxons/genética , Regulação da Expressão Gênica , Lipase Lipoproteica/metabolismo , Linfadenopatia/genética , Linfadenopatia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Triglicerídeos/sangueRESUMO
Angiopoietin-like (ANGPTL) 8 is a secreted inhibitor of LPL, a key enzyme in plasma triglyceride metabolism. It was previously reported that ANGPTL8 requires another member of the ANGPTL family, ANGPTL3, to act on LPL. ANGPTL3, much like ANGPTL4, is a physiologically relevant regulator of LPL activity, which causes irreversible inactivation of the enzyme. Here, we show that ANGPTL8 can form complexes with either ANGPTL3 or ANGPTL4 when the proteins are refolded together from their denatured states. In contrast to the augmented inhibitory effect of the ANGPTL3/ANGPTL8 complex on LPL activity, the ANGPTL4/ANGPTL8 complex is less active compared with ANGPTL4 alone. In our experiments, all three members of the ANGPTL family use the same mechanism to inactivate LPL, which involves dissociation of active dimeric LPL to monomers. This inactivation can be counteracted by the presence of glycosylphosphatidylinositol-anchored HDL binding protein 1, the endothelial LPL transport protein previously known to protect LPL from spontaneous and ANGPTL4-catalyzed inactivation. Our data demonstrate that ANGPTL8 may function as an important metabolic switch, by forming complexes with ANGPTL3, or with ANGPTL4, in order to direct the flow of energy from triglycerides in blood according to the needs of the body.
Assuntos
Proteínas Semelhantes a Angiopoietina/biossíntese , Lipase Lipoproteica/metabolismo , Hormônios Peptídicos/biossíntese , Proteína 8 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina/genética , Proteínas Semelhantes a Angiopoietina/isolamento & purificação , Humanos , Hormônios Peptídicos/genética , Hormônios Peptídicos/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Activity of lipoprotein lipase (LPL) is high in mouse kidney, but the reason is poorly understood. The aim was to characterize localization, regulation, and function of LPL in kidney of C57BL/6J mice. We found LPL mainly in proximal tubules, localized inside the tubular epithelial cells, under all conditions studied. In fed mice, some LPL colocalized with the endothelial markers CD31 and GPIHBP1 and could be removed by perfusion with heparin, indicating a vascular location. The role of angiopoietin-like protein 4 (ANGPTL4) for nutritional modulation of LPL activity was studied in wild-type and Angptl4-/- mice. In Angptl4-/- mice, kidney LPL activity remained high in fasted animals, indicating that ANGPTL4 is involved in suppression of LPL activity on fasting, like in adipose tissue. The amount of ANGPTL4 protein in kidney was low, and the protein appeared smaller in size, compared with ANGPTL4 in heart and adipose tissue. To study the influence of obesity, mice were challenged with high-fat diet for 22 wk, and LPL was studied after an overnight fast compared with fasted mice given food for 3 h. High-fat diet caused blunting of the normal adaptation of LPL activity to feeding/fasting in adipose tissue, but in kidneys this adaptation was lost only in male mice. LPL activity increases to high levels in mouse kidney after feeding, but as no difference in uptake of chylomicron triglycerides in kidneys is found between fasted and fed states, our data confirm that LPL appears to have a minor role for lipid uptake in this organ.
Assuntos
Dieta Hiperlipídica , Rim/metabolismo , Lipase Lipoproteica/metabolismo , Obesidade/metabolismo , Tecido Adiposo/metabolismo , Proteína 4 Semelhante a Angiopoietina/genética , Proteína 4 Semelhante a Angiopoietina/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Knockout , Estado Nutricional , Fatores SexuaisRESUMO
The intravascular processing of triglyceride-rich lipoproteins depends on lipoprotein lipase (LPL) and GPIHBP1, a membrane protein of endothelial cells that binds LPL within the subendothelial spaces and shuttles it to the capillary lumen. In the absence of GPIHBP1, LPL remains mislocalized within the subendothelial spaces, causing severe hypertriglyceridemia (chylomicronemia). The N-terminal domain of GPIHBP1, an intrinsically disordered region (IDR) rich in acidic residues, is important for stabilizing LPL's catalytic domain against spontaneous and ANGPTL4-catalyzed unfolding. Here, we define several important properties of GPIHBP1's IDR. First, a conserved tyrosine in the middle of the IDR is posttranslationally modified by O-sulfation; this modification increases both the affinity of GPIHBP1-LPL interactions and the ability of GPIHBP1 to protect LPL against ANGPTL4-catalyzed unfolding. Second, the acidic IDR of GPIHBP1 increases the probability of a GPIHBP1-LPL encounter via electrostatic steering, increasing the association rate constant (kon) for LPL binding by >250-fold. Third, we show that LPL accumulates near capillary endothelial cells even in the absence of GPIHBP1. In wild-type mice, we expect that the accumulation of LPL in close proximity to capillaries would increase interactions with GPIHBP1. Fourth, we found that GPIHBP1's IDR is not a key factor in the pathogenicity of chylomicronemia in patients with the GPIHBP1 autoimmune syndrome. Finally, based on biophysical studies, we propose that the negatively charged IDR of GPIHBP1 traverses a vast space, facilitating capture of LPL by capillary endothelial cells and simultaneously contributing to GPIHBP1's ability to preserve LPL structure and activity.
Assuntos
Células Endoteliais/metabolismo , Lipase Lipoproteica/metabolismo , Receptores de Lipoproteínas/metabolismo , Proteína 4 Semelhante a Angiopoietina/química , Proteína 4 Semelhante a Angiopoietina/genética , Proteína 4 Semelhante a Angiopoietina/metabolismo , Animais , Células Endoteliais/patologia , Humanos , Hiperlipoproteinemia Tipo I/genética , Hiperlipoproteinemia Tipo I/metabolismo , Hiperlipoproteinemia Tipo I/patologia , Lipase Lipoproteica/química , Lipase Lipoproteica/genética , Camundongos , Ligação Proteica , Domínios Proteicos , Receptores de Lipoproteínas/química , Receptores de Lipoproteínas/genética , Tirosina/química , Tirosina/genética , Tirosina/metabolismoRESUMO
OBJECTIVE: Tissue macrophages induce and perpetuate proinflammatory responses, thereby promoting metabolic and cardiovascular disease. Lipoprotein lipase (LpL), the rate-limiting enzyme in blood triglyceride catabolism, is expressed by macrophages in atherosclerotic plaques. We questioned whether LpL, which is also expressed in the bone marrow (BM), affects circulating white blood cells and BM proliferation and modulates macrophage retention within the artery. APPROACH AND RESULTS: We characterized blood and tissue leukocytes and inflammatory molecules in transgenic LpL knockout mice rescued from lethal hypertriglyceridemia within 18 hours of life by muscle-specific LpL expression (MCKL0 mice). LpL-deficient mice had ≈40% reduction in blood white blood cell, neutrophils, and total and inflammatory monocytes (Ly6C/Ghi). LpL deficiency also significantly decreased expression of BM macrophage-associated markers (F4/80 and TNF-α [tumor necrosis factor α]), master transcription factors (PU.1 and C/EBPα), and colony-stimulating factors (CSFs) and their receptors, which are required for monocyte and monocyte precursor proliferation and differentiation. As a result, differentiation of macrophages from BM-derived monocyte progenitors and monocytes was decreased in MCKL0 mice. Furthermore, although LpL deficiency was associated with reduced BM uptake and accumulation of triglyceride-rich particles and macrophage CSF-macrophage CSF receptor binding, triglyceride lipolysis products (eg, linoleic acid) stimulated expression of macrophage CSF and macrophage CSF receptor in BM-derived macrophage precursor cells. Arterial macrophage numbers decreased after heparin-mediated LpL cell dissociation and by genetic knockout of arterial LpL. Reconstitution of LpL-expressing BM replenished aortic macrophage density. CONCLUSIONS: LpL regulates peripheral leukocyte levels and affects BM monocyte progenitor differentiation and aortic macrophage accumulation.
Assuntos
Aorta/enzimologia , Doenças da Aorta/enzimologia , Aterosclerose/enzimologia , Hiperlipoproteinemia Tipo I/enzimologia , Lipase Lipoproteica/deficiência , Macrófagos/enzimologia , Monócitos/enzimologia , Células Progenitoras Mieloides/enzimologia , Mielopoese , Animais , Aorta/patologia , Doenças da Aorta/sangue , Doenças da Aorta/genética , Doenças da Aorta/patologia , Aterosclerose/sangue , Aterosclerose/genética , Aterosclerose/patologia , Proliferação de Células , Citocinas/metabolismo , Dieta Hiperlipídica , Modelos Animais de Doenças , Hiperlipoproteinemia Tipo I/sangue , Hiperlipoproteinemia Tipo I/genética , Hiperlipoproteinemia Tipo I/patologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Lipase Lipoproteica/genética , Macrófagos/patologia , Camundongos Knockout , Monócitos/patologia , Células Progenitoras Mieloides/patologia , Transdução de Sinais , Triglicerídeos/metabolismoRESUMO
PURPOSE: We studied effects of diet-induced postmenopausal weight loss on gene expression and activity of proteins involved in lipogenesis and lipolysis in adipose tissue. METHODS: Fifty-eight postmenopausal women with overweight (BMI 32.5 ± 5.5) were randomized to eat an ad libitum Paleolithic-type diet (PD) aiming for a high intake of protein and unsaturated fatty acids or a prudent control diet (CD) for 24 months. Anthropometry, plasma adipokines, gene expression of proteins involved in fat metabolism in subcutaneous adipose tissue (SAT) and lipoprotein lipase (LPL) activity and mass in SAT were measured at baseline and after 6 months. LPL mass and activity were also measured after 24 months. RESULTS: The PD led to improved insulin sensitivity (P < 0.01) and decreased circulating triglycerides (P < 0.001), lipogenesis-related factors, including LPL mRNA (P < 0.05), mass (P < 0.01), and activity (P < 0.001); as well as gene expressions of CD36 (P < 0.05), fatty acid synthase, FAS (P < 0.001) and diglyceride acyltransferase 2, DGAT2 (P < 0.001). The LPL activity (P < 0.05) and gene expression of DGAT2 (P < 0.05) and FAS (P < 0.05) were significantly lowered in the PD group versus the CD group at 6 months and the LPL activity (P < 0.05) remained significantly lowered in the PD group compared to the CD group at 24 months. CONCLUSIONS: Compared to the CD, the PD led to a more pronounced reduction of lipogenesis-promoting factors in SAT among postmenopausal women with overweight. This could have mediated the favorable metabolic effects of the PD on triglyceride levels and insulin sensitivity.
Assuntos
Dieta Paleolítica , Lipogênese , Sobrepeso/sangue , Pós-Menopausa , Gordura Subcutânea/metabolismo , Adipocinas/sangue , Idoso , Antropometria , Diacilglicerol O-Aciltransferase/genética , Diacilglicerol O-Aciltransferase/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Lipase Lipoproteica/metabolismo , Pessoa de Meia-Idade , Triglicerídeos/sangue , Redução de Peso , Receptor fas/genética , Receptor fas/metabolismoRESUMO
Lipoprotein lipase (LPL) hydrolyzes lipids in plasma lipoproteins so that the fatty acids can be taken up and used by cells. The activity of LPL changes rapidly in response to changes in nutrition, physical activity and other conditions. Angiopoietin-like protein 4 (ANGPTL4) is an important controller of LPL activity. Both LPL and ANGPTL4 are produced and secreted by adipocytes. When the transcription blocker Actinomycin D was added to cultures of 3T3-L1 adipocytes, LPL activity in the medium increased several-fold. LPL mRNA decreased moderately during 5h, while ANGPTL4 mRNA and protein declined rapidly, explaining that LPL activity was increased. TNF-α is known to reduce LPL activity in adipose tissue. We have shown that TNF-α increased ANGPTL4 both at the mRNA and protein level. Expression of ANGPTL4 is known to be under control of Foxo1. Use of the Foxo1-specific inhibitor AS1842856, or knockdown of ANGPTL4 by RNAi, resulted in increased LPL activity in the medium. Both with ActD and with the Foxo1 inhibitor the cells became unresponsive to TNF-α. This study shows that TNF-α, by a Foxo1 dependent pathway, increases the transcription of ANGPTL4 which is secreted by the cells and causes inactivation of LPL.
Assuntos
Adipócitos/metabolismo , Angiopoietinas/biossíntese , Proteína Forkhead Box O1/metabolismo , Lipase Lipoproteica/biossíntese , Fator de Necrose Tumoral alfa/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Proteína 4 Semelhante a Angiopoietina , Angiopoietinas/genética , Animais , Dactinomicina/administração & dosagem , Proteína Forkhead Box O1/antagonistas & inibidores , Regulação da Expressão Gênica/efeitos dos fármacos , Lipase Lipoproteica/genética , Camundongos , Quinolonas/administração & dosagem , Interferência de RNA , RNA Mensageiro/biossíntese , Ativação Transcricional/efeitos dos fármacos , Fator de Necrose Tumoral alfa/administração & dosagemRESUMO
Plasma apoC-III levels correlate with triglyceride (TG) levels and are a strong predictor of CVD outcomes. ApoC-III elevates TG in part by inhibiting LPL. ApoC-III likely inhibits LPL by competing for lipid binding. To probe this, we used oil-drop tensiometry to characterize binding of six apoC-III variants to lipid/water interfaces. This technique monitors the dependence of lipid binding on surface pressure, which increases during TG hydrolysis by LPL. ApoC-III adsorption increased surface pressure by upward of 18 mN/m at phospholipid/TG/water interfaces. ApoC-III was retained to high pressures at these interfaces, desorbing at 21-25 mN/m. Point mutants, which substituted alanine for aromatic residues, impaired the lipid binding of apoC-III. Adsorption and retention pressures decreased by 1-6 mN/m in point mutants, with the magnitude determined by the location of alanine substitutions. Trp42 was most critical to mediating lipid binding. These results strongly correlate with our previous results, linking apoC-III point mutants to increased LPL binding and activity at lipid surfaces. We propose that aromatic residues in the C-terminal half of apoC-III mediate binding to TG-rich lipoproteins. Increased apoC-III expression in the hypertriglyceridemic state allows apoC-III to accumulate on lipoproteins and inhibit LPL by preventing binding and/or access to substrate.
Assuntos
Apolipoproteína C-II/química , Apolipoproteína C-II/metabolismo , Metabolismo dos Lipídeos , Lipase Lipoproteica/antagonistas & inibidores , Adsorção , Sequência de Aminoácidos , Apolipoproteína C-II/genética , Humanos , Mutação , Relação Estrutura-Atividade , Triglicerídeos/metabolismoRESUMO
Angiopoietin-like protein 4 (ANGPTL4) is suggested to be a master regulator of plasma triglyceride metabolism. Our aim was to study whether the previously reported high levels of ANGPTL4 detected in serum from patients with rheumatoid arthritis (RA) by ELISA was due to any specific molecular form of this protein (oligomers, monomers or fragments). ANGPTL4 levels were first determined in serum from 68 RA patients and 43 age and sex matched control subjects and the mean values differed by a factor of 5.0. Then, ANGPTL4 was analyzed after size exclusion chromatography (SEC) of serum samples. With serum from one of the RA patients with high levels of ANGPTL4, the dominant reactivity was found in fractions corresponding to high-molecular weight proteins. In addition, a minor peak of reactivity eluting late from the column was found both in the patient and in controls. By the use of HeteroBlock®, and by careful selection of antibodies, we documented non-specific reactions for ANGPTL4 in 39% of samples from the RA patients, most likely due to cross-reactivity of the antibodies with rheumatoid factor (RF). The corresponding figure for control subjects was 6.3%. After corrections for non-specific reactions, the mean level of ANGPTL4 in serum from RA patients was still significantly higher than in control individuals (mean levels were 101±62 and 67±39 ng/ml respectively, P = 0.02). We re-analyzed samples from our previously published studies on ANGPL4 levels in patients on hemodialysis and patients with diabetes type 2. These samples did not show false positive reactions. The levels of ANGPTL4 were comparable to those detected previously.
Assuntos
Angiopoietinas/sangue , Artrite Reumatoide/sangue , Autoanticorpos/imunologia , Angiopoietinas/imunologia , Artrite Reumatoide/imunologia , Cromatografia em Gel , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
LPL hydrolyzes triglycerides in plasma lipoproteins. Due to the complex regulation mechanism, it has been difficult to mimic the physiological conditions under which LPL acts in vitro. We demonstrate that isothermal titration calorimetry (ITC), using human plasma as substrate, overcomes several limitations of previously used techniques. The high sensitivity of ITC allows continuous recording of the heat released during hydrolysis. Both initial rates and kinetics for complete hydrolysis of plasma lipids can be studied. The heat rate was shown to correspond to the release of fatty acids and was linearly related to the amount of added enzyme, either purified LPL or postheparin plasma. Addition of apoC-III reduced the initial rate of hydrolysis by LPL, but the inhibition became less prominent with time when the lipoproteins were triglyceride poor. Addition of angiopoietin-like protein (ANGPTL)3 or ANGPTL4 caused reduction of the activity of LPL via a two-step mechanism. We conclude that ITC can be used for quantitative measurements of LPL activity and interactions under in vivo-like conditions, for comparisons of the properties of plasma samples from patients and control subjects as substrates for LPL, as well as for testing of drug candidates developed with the aim to affect the LPL system.
Assuntos
Calorimetria , Ácidos Graxos/sangue , Lipólise/efeitos dos fármacos , Lipase Lipoproteica/sangue , Adulto , Proteína 3 Semelhante a Angiopoietina , Proteína 4 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/administração & dosagem , Animais , Apolipoproteína C-III/administração & dosagem , Bovinos , Feminino , Voluntários Saudáveis , Humanos , Hidrólise , Cinética , Lipoproteínas VLDL/sangue , Masculino , Triglicerídeos/sangueRESUMO
Lipoprotein lipase (LPL) undergoes spontaneous inactivation via global unfolding and this unfolding is prevented by GPIHBP1 (Mysling et al., 2016). We now show: (1) that ANGPTL4 inactivates LPL by catalyzing the unfolding of its hydrolase domain; (2) that binding to GPIHBP1 renders LPL largely refractory to this inhibition; and (3) that both the LU domain and the intrinsically disordered acidic domain of GPIHBP1 are required for this protective effect. Genetic studies have found that a common polymorphic variant in ANGPTL4 results in lower plasma triglyceride levels. We now report: (1) that this ANGPTL4 variant is less efficient in catalyzing the unfolding of LPL; and (2) that its Glu-to-Lys substitution destabilizes its N-terminal α-helix. Our work elucidates the molecular basis for regulation of LPL activity by ANGPTL4, highlights the physiological relevance of the inherent instability of LPL, and sheds light on the molecular defects in a clinically relevant variant of ANGPTL4.
Assuntos
Proteína 4 Semelhante a Angiopoietina/metabolismo , Lipase Lipoproteica/metabolismo , Dobramento de Proteína , Receptores de Lipoproteínas/metabolismo , Proteína 4 Semelhante a Angiopoietina/genética , Lipase Lipoproteica/química , Espectrometria de Massas , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Domínios Proteicos , Mapeamento de Interação de ProteínasRESUMO
BACKGROUND: The relationship of conventional cardiovascular risk factors (age, gender, ethnicity, diabetes, dyslipidaemia, hypertension, obesity, exercise, and the number of risk factors) to coronary artery calcification (CAC) presence and extent has never before been assessed in a systematic review and meta-analysis. METHODS: We included only English language studies that assessed at least three conventional risk factors apart from age, gender, and ethnicity, but excluded studies in which all patients had another confirmed condition such as renal disease. RESULTS: In total, 10 studies, comprising 15,769 patients, were investigated in the systematic review and seven studies, comprising 12,682 patients, were included in the meta-analysis, which demonstrated the importance of diabetes and hypertension as predictors of CAC presence and extent, with age also predicting CAC presence. Male gender, dyslipidaemia, family history of coronary artery disease, obesity, and smoking were overall not predictive of either CAC presence or extent, despite dyslipidaemia being a key risk factor for coronary artery disease (CAD). CONCLUSION: Diabetes and hypertension consistently predict the presence and extent of CAC in symptomatic patients.
Assuntos
Doença da Artéria Coronariana/epidemiologia , Diabetes Mellitus/epidemiologia , Hipertensão/epidemiologia , Calcificação Vascular/epidemiologia , Idoso , Vasos Coronários/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
In humans, genetic variation of sortilin-related receptor, L(DLR class) A repeats containing (SORL1), which encodes the intracellular sorting receptor SORLA, is a major genetic risk factor for familial and sporadic forms of Alzheimer's disease. Recent GWAS analysis has also associated SORL1 with obesity in humans and in mouse models, suggesting that this receptor may play a role in regulating metabolism. Here, using mouse models with genetic loss or tissue-specific overexpression of SORLA as well as data from obese human subjects, we observed a gene-dosage effect that links SORLA expression to obesity and glucose tolerance. Overexpression of human SORLA in murine adipose tissue blocked hydrolysis of triacylglycerides and caused excessive adiposity. In contrast, Sorl1 gene inactivation in mice accelerated breakdown of triacylglycerides in adipocytes and protected animals from diet-induced obesity. We then identified the underlying molecular mechanism whereby SORLA promotes insulin-induced suppression of lipolysis in adipocytes. Specifically, we determined that SORLA acts as a sorting factor for the insulin receptor (IR) that redirects internalized receptor molecules from endosomes to the plasma membrane, thereby enhancing IR surface expression and strengthening insulin signal reception in target cells. Our findings provide a molecular mechanism for the association of SORL1 with human obesity and confirm a genetic link between neurodegeneration and metabolism that converges on the receptor SORLA.