RESUMEN
BACKGROUND: Atherosclerotic cardiovascular disease is the main cause of mortality worldwide and is strongly influenced by circulating low-density lipoprotein (LDL) cholesterol levels. Only a few genes causally related to plasma LDL cholesterol levels have been identified so far, and only 1 gene, ANGPTL3, has been causally related to combined hypocholesterolemia. Here, our aim was to elucidate the genetic origin of an unexplained combined hypocholesterolemia inherited in 4 generations of a French family. METHODS: Using next-generation sequencing, we identified a novel dominant rare variant in the LIPC gene, encoding for hepatic lipase, which cosegregates with the phenotype. We characterized the impact of this LIPC-E97G variant on circulating lipid and lipoprotein levels in family members using nuclear magnetic resonance-based lipoprotein profiling and lipidomics. To uncover the mechanisms underlying the combined hypocholesterolemia, we used protein homology modeling, measured triglyceride lipase and phospholipase activities in cell culture, and studied the phenotype of APOE*3.Leiden.CETP mice after LIPC-E97G overexpression. RESULTS: Family members carrying the LIPC-E97G variant had very low circulating levels of LDL cholesterol and high-density lipoprotein cholesterol, LDL particle numbers, and phospholipids. The lysophospholipids/phospholipids ratio was increased in plasma of LIPC-E97G carriers, suggestive of an increased lipolytic activity on phospholipids. In vitro and in vivo studies confirmed that the LIPC-E97G variant specifically increases the phospholipase activity of hepatic lipase through modification of an evolutionarily conserved motif that determines substrate access to the hepatic lipase catalytic site. Mice overexpressing human LIPC-E97G recapitulated the combined hypocholesterolemic phenotype of the family and demonstrated that the increased phospholipase activity promotes catabolism of triglyceride-rich lipoproteins by different extrahepatic tissues but not the liver. CONCLUSIONS: We identified and characterized a novel rare variant in the LIPC gene in a family who presents with dominant familial combined hypocholesterolemia. This gain-of-function variant makes LIPC the second identified gene, after ANGPTL3, causally involved in familial combined hypocholesterolemia. Our mechanistic data highlight the critical role of hepatic lipase phospholipase activity in LDL cholesterol homeostasis and suggest a new LDL clearance mechanism.
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Mutación con Ganancia de Función , Lipasa , Proteína 3 Similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina/genética , Animales , HDL-Colesterol , LDL-Colesterol , Humanos , Lipasa/genética , Lipoproteínas , Ratones , Fosfolipasas/genéticaRESUMEN
Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes lysosomal degradation of the LDL receptor and is a key regulator of cholesterol metabolism. After the liver, the small intestine is the second organ that highly expresses PCSK9. However, the small intestine's ability to secrete PCSK9 remains a matter of debate. While liver-specific PCSK9-deficient mice present no PCSK9 in systemic blood, human intestinal Caco-2 cells can actively secrete PCSK9. This raises the possibility for active intestinal secretion via the portal blood. Here, we aimed to determine whether enterocytes can secrete PCSK9 using in vitro, ex vivo, and in vivo approaches. We first observed that PCSK9 secretion from Caco-2 cells was biphasic and dependent on Caco-2 maturation status. Transcriptional analysis suggested that this transient reduction in PCSK9 secretion might be due to loss of SREBP2-mediated transcription of PCSK9. Consistently, PCSK9 secretion was not detected ex vivo in human or mouse intestinal biopsies mounted in Ussing chambers. Finally, direct comparison of systemic versus portal blood PCSK9 concentrations in WT or liver-specific PCSK9-deficient mice confirmed the inability of the small intestine to secrete PCSK9 into the portal compartment. Altogether, our data demonstrate that mature enterocytes do not secrete PCSK9 and reinforce the central role of the liver in the regulation of the concentration of circulating PCSK9 and consequently of cellular LDL receptors.
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Proproteína Convertasa 9/metabolismo , Animales , Células CACO-2 , Diferenciación Celular , Células Cultivadas , Humanos , Intestino Delgado/citología , Intestino Delgado/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proproteína Convertasa 9/sangre , Proproteína Convertasa 9/deficienciaAsunto(s)
Compuestos de Bencidrilo , Modelos Animales de Enfermedad , Glucósidos , Lipogénesis , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Animales , Compuestos de Bencidrilo/uso terapéutico , Compuestos de Bencidrilo/farmacología , Glucósidos/uso terapéutico , Glucósidos/farmacología , Lipogénesis/efectos de los fármacos , Ratones , Inhibidores del Cotransportador de Sodio-Glucosa 2/uso terapéutico , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Hígado Graso/tratamiento farmacológico , Hígado Graso/metabolismo , Masculino , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/complicacionesRESUMEN
OBJECTIVE: The objective of this study was to compare the general and metabolic impact of single-anastomosis duodeno-ileal bypass with sleeve gastrectomy (SADI-S) with Roux-en-Y gastric bypass (RYGB) in an obese (ob/ob) mouse model. METHODS: 10-week-old male ob/ob mice underwent either SADI-S, RYGB, or laparotomy surgery (Sham group). General and metabolic parameters were assessed during a 5-week period thereafter. RESULTS: SADI-S induced a deeper weight loss ([mean ± SEM] -41.2% ± 3.3%) than RYGB (-5.6% ± 3.5%, p < 0.001) compared with the Sham group (+6.3% ± 1.0%, p < 0.05). A significant food restriction was observed after SADI-S only (-31%, 117.4 ± 10.3 g vs. 170.2 ± 5.2 g of food at day 35 in Sham group mice, p < 0.001). Random-fed glycemia and glucose tolerance were more improved after SADI-S than RYGB. SADI-S decreased plasma cholesterol concentration by 60% (0.49 ± 0.04 g/L vs. 1.40 ± 0.10 g/L in the Sham group at day 35, p < 0.01), significantly more than RYGB (1.04 ± 0.14 g/L, p = 0.018). Plasma sitosterol/cholesterol and campesterol/cholesterol ratios were decreased after SADI-S, suggesting a reduced intestinal cholesterol absorption. SADI-S increased exogenous plasma cholesterol-D7 clearance and fecal elimination, also indicating an increased plasma cholesterol excretion. Studying a pair-fed group demonstrated that calorie restriction alone did not explain the beneficial impact of SADI-S. CONCLUSIONS: SADI-S is associated with a greater improvement in lipid and glucose homeostasis than RYGB in ob/ob mice.
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Derivación Gástrica , Obesidad Mórbida , Animales , Masculino , Ratones , Colesterol , Gastrectomía , Glucosa , Homeostasis , Lípidos , Obesidad Mórbida/cirugía , Estudios Retrospectivos , Ratones ObesosRESUMEN
The Proprotein Convertase Subtilisin Kexin of type 9 (PCSK9) has been identified in 2003 as the third gene involved in familial hypercholesterolemia. PCSK9 binds to the membrane low-density lipoprotein receptor (LDLR) and promotes its cellular internalization and lysosomal degradation. Beyond this canonical role, PCSK9 was recently described to be involved in several immune responses. However, to date, the contribution of PCSK9 in food allergy remains unknown. Here, we showed that Pcsk9 deficiency or pharmacological inhibition of circulating PCSK9 with a specific monoclonal antibody (m-Ab) protected mice against symptoms of gliadin-induced-food allergy, such as increased intestinal transit time and ear oedema. Furthermore, specific PCSK9 inhibition during the elicitation steps of allergic process was sufficient to ensure anti-allergic effects in mice. Interestingly, the protective effect of PCSK9 inhibition against food allergy symptoms was independent of the LDLR as PCSK9 inhibitors remained effective in Ldlr deficient mice. In vitro, we showed that recombinant gain of function PCSK9 (PCSK9 D374Y) increased the percentage of mature bone marrow derived dendritic cells (BMDCs), promoted naïve T cell proliferation and potentiated the gliadin induced basophils degranulation. Altogether, our data demonstrate that PCSK9 inhibition is protective against gliadin induced food allergy in a LDLR-independent manner.
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Obesity is a major health issue worldwide. As a response, bariatric surgeries have emerged to treat obesity and its related comorbidities (e.g., diabetes mellitus, dyslipidemia, non-alcoholic steatohepatitis, cardiovascular events, and cancers) through restrictive and malabsorptive mechanisms. Understanding the mechanisms by which these procedures allow such improvements often require their transposition into animals, especially in mice, because of the ease of generating genetically modified animals. Recently, the single-anastomosis duodeno-ileal bypass with sleeve gastrectomy (SADI-S) has emerged as a procedure that uses both restrictive and malabsorptive effects, which is being used as an alternative to gastric bypass in case of major obesity. Thus far, this procedure has been associated with strong metabolic improvements, which has led to a marked increase in its use in daily clinical practice. However, the mechanisms underlying these metabolic effects have been poorly studied as a result of a lack of animal models. In this article, we present a reliable and reproducible model of SADI-S in mice, with a special focus on perioperative management. The description and use of this new rodent model will be helpful for the scientific community to better understand the molecular, metabolic, and structural changes induced by the SADI-S and to better define the surgical indications for clinical practice.
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Derivación Gástrica , Obesidad Mórbida , Ratones , Animales , Obesidad Mórbida/cirugía , Derivación Gástrica/métodos , Obesidad/cirugía , Obesidad/complicaciones , Duodeno/cirugía , Gastrectomía/métodos , Anastomosis Quirúrgica/métodos , Estudios RetrospectivosRESUMEN
PCSK9 promotes the lysosomal degradation of cell surface LDL receptor (LDLR). We analyzed how excess LDLR generated by PCSK9 deficiency is differently handled in male and female mice to possibly unveil the mechanism leading to the lower efficacy of PCSK9 mAb on LDL-cholesterol levels in women. Analysis of intact or ovariectomized PCSK9 knockout (KO) mice supplemented with placebo or 17ß-estradiol (E2) demonstrated that female, but not male mice massively shed the soluble ectodomain of the LDLR in the plasma. Liver-specific PCSK9 KO or alirocumab-treated WT mice exhibit the same pattern. This shedding is distinct from the basal one and is inhibited by ZLDI-8, a metalloprotease inhibitor pointing at ADAM10/ADAM17. In PCSK9 KO female mice, ZLDI-8 raises by 80â¯% the LDLR liver content in a few hours. This specific shedding is likely cholesterol-dependent: it is prevented in PCSK9 KO male mice that exhibit low intra-hepatic cholesterol levels without activating SREBP-2, and enhanced by mevalonate or high cholesterol feeding, or by E2 known to stimulate cholesterol synthesis via the estrogen receptor-α. Liver transcriptomics demonstrates that critically low liver cholesterol in ovariectomized female or knockout male mice also hampers the cholesterol-dependent G2/M transition of the cell cycle. Finally, higher levels of shed LDLR were measured in the plasma of women treated with PCSK9 mAb. PCSK9 knockout female mice hormonally sustain cholesterol synthesis and shed excess LDLR, seemingly like women. In contrast, male mice rely on high surface LDLR to replenish their stocks, despite 80â¯% lower circulating LDL.