Objective-Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a central player in the regulation of cholesterol homeostasis, increasing the low-density lipoprotein (LDL) receptor degradation. Our study aimed at exploring the pathogenic consequences in vivo and in vitro of a PCSK9 prodomain mutation found in a family with hypobetalipoproteinemia (FHBL). Methods and Results-A white 49-year-old diabetic man had profound FBHL (LDLC: 16 mg/dL) whereas his daughter and sister displayed a milder phenotype (LDLC 44 mg/dL and 57 mg/dL, respectively), all otherwise healthy with a normal liver function. A monoallelic PCSK9 double-mutant R104C/V114A cosegregated with FBHL, with no mutation found at other FHBL-causing loci. A dose-effect was also found in FBHL relatives for plasma APOB and PCSK9 (very-low to undetectable in proband, Ϸ50% decreased in sister and daughter) and LDL catabolic rate (256% and 88% increased in proband and daughter). Transient transfection in hepatocytes showed severely impaired processing and secretion of the double mutant which acted as a dominant negative over secretion of wild-type PCSK9. Conclusion-These results show that heterozygous PCSK9 missense mutations may associate with profound hypobetalipoproteinemia and constitute the first direct evidence in human that decrease of plasma LDLC concentrations associated to PCSK9 LOF mutations are attributable to an increased clearance rate of LDL. Key Words: PCSK9 Ⅲ LDL Ⅲ mutation Ⅲ hypobetalipoproteinemia H ypobetalipoproteinemia (HBL) refers to a heterogeneous group of monogenic disorders characterized by very low plasma concentrations of low-density lipoprotein cholesterol (LDLC) and apolipoprotein B (apoB) (ie, Ͻ5 percentile of the distribution in the population; for review see 1 ). HBL includes 3 inherited disorders: (1) familial hypobetalipoproteinemia (FHBL; OMIM 107730), (2) abetalipoproteinemia (ABL; OMIM 200100), and (3) chylomicron retention disease (CRD; OMIM 246700). The frequency of subjects with heterozygous FHBL has been estimated to be 1:500/1:1000. 2 FHBL heterozygotes are often asymptomatic or express mild clinical manifestations such as fatty liver disease and intestinal fat malabsorption. 3 FHBL are often caused by APOB gene mutations. 1,4 However, a substantial number of FHBL (varying to 36% 1 to 56% 5 in the literature) do not harbor apoB mutations.In the last 5 years, proprotein convertase subtilisin/kexin 9 (PCSK9) has emerged as a crucial modulator of cholesterol metabolism. 6 PCSK9 is primarily expressed in the liver and the intestine. PCSK9 inhibits the LDL receptor (LDLR) pathway in a posttranscriptional manner. 6 In human, PCSK9 was initially reported as the third gene causing autosomal dominant hypercholesterolemia, in addition to LDLR and APOB mutations. 7 Indeed, PCSK9 gain-of-function (GOF) mutations lead to increased plasma LDLC levels and premature atherosclerosis. 7,8 In contrast, PCSK9 loss-of-function (LOF) mutations are associated with low LDLC levels and protection against coronary diseases. 9,10 To dat...

The cell death-inducing DFFA-like effector c (CIDEC; also known in rodents as FSP27 or fat-specific protein 27) is a lipid droplet-associated protein that promotes intracellular triglyceride storage. CIDEC/Fsp27 is highly expressed in adipose tissue but undetectable in normal liver. Its hepatic expression, however, rises during fasting or under genetic or diet-induced hepatosteatosis in both mice and patients. Herein, we demonstrate that CIDEC/Fsp27 is a direct transcriptional target of the nuclear receptor PPARα (peroxisome proliferator activated receptor alpha) in both mouse and human hepatocytes, and that preventing Fsp27 induction accelerates PPARα-stimulated fatty acid oxidation. We show that adenoviral-mediated silencing of hepatic Fsp27 abolishes fasting-induced liver steatosis in the absence of changes in plasma lipids. Finally, we report that anti-Fsp27 shRNA and PPARα agonists synergize to ameliorate hepatosteatosis in mice fed a high fat diet. Together, our data highlight the physiological importance of CIDEC/Fsp27 in triglyceride homeostasis under both physiological and pathological liver steatosis. Our results also suggest that patients taking fibrates likely have elevated levels of hepatic CIDEC, which may limit the efficient mobilization and catabolism of hepatic triglycerides.

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Objectives-Proprotein convertase subtilisin kexin type 9 (PCSK9) is a natural inhibitor of the low-density lipoprotein receptor, and its deficiency in humans results in low plasma LDL-cholesterol and protection against cardiovascular disease. We explored whether PCSK9 expression impacts postprandial triglyceridemia, another important cardiovascular risk factor. Methods and Results-Real-time PCR and confocal microscopy were used to show that PCSK9 is expressed throughout the entire small intestine and in human enterocytes. On olive oil gavage, PCSK9-deficient mice showed a dramatically decreased postprandial triglyceridemia compared with their wild-type littermates. Lymph analysis revealed that intestinal TG output is not quantitatively modified by PCSK9 deletion. However, PCSK9 Ϫ/Ϫ mice present with a significant reduction of lymphatic apoB secretion compared to PCSK9 ϩ/ϩ mice. Modulating PCSK9 expression in polarized CaCo-2 cells confirmed the relationship between PCSK9 and apoB secretion; PCSK9 Ϫ/Ϫ mice consistently secrete larger TG-rich lipoprotein than wild-type littermates. Finally, kinetic studies showed that PCSK9-deficient mice have an increased ability to clear chylomicrons compared to wild-type littermates. G ain-of-function mutations affecting proprotein convertase subtilisin/kexin type 9 (PCSK9) are associated with autosomal dominant hypercholesterolemia and premature atherosclerosis. 1 It is now established that PCSK9 is a natural inhibitor of the low-density lipoprotein receptor (LDLr), acting posttranscriptionally. 2 Circulating PCSK9 binds to the EGF-A extracellular domain of the hepatic LDLr and prevents its recycling to the cell surface. 3 A breakthrough study reported that blacks harboring PCSK9 loss-of-function mutations had an 88% risk reduction for coronary heart disease. 4 Although the lower concentrations of LDL cholesterol over one's lifetime is suggested to be the main reason for this very high level of protection, we hypothesized that PCSK9 deficiency might also affect other risk factors. Low levels of HDL cholesterol (HDL-c) and elevated nonfasted triglycerides (TG) levels are associated with increased risk for cardiovascular disease. No association between PCSK9 loss-of-function mutations and plasma HDL-c levels has been noticed. 4,5 However, to our knowledge, nonfasting plasma TG were not measured in PCSK9-deficient individuals. They are an important contributor to the risk for cardiovascular disease. 6,7 Rashid et al reported the phenotype of PCSK9-deficient mice (PCSK9 Ϫ/Ϫ ) mice and observed that they exhibit lower plasma cholesterol levels (Ϫ50%) and are hypersensitive to statins. 8 The potential role of PCSK9 in the intestine in mice and humans remains unexplored. Conclusion-TheseDuring the postprandial period, dietary lipids are actively absorbed into the enterocyte. After intracellular reesterification, long chain fatty acids and cholesterol esters are associated with phospholipids and apolipoproteins, particularly apoB48, to produce TG-rich lipoproteins, mainly chy...

Understanding the hepatic regenerative process has clinical interest, since the effectiveness of many treatments for chronic liver diseases is conditioned by an efficient liver regeneration. Experimental evidence points to the need of a temporal coordination between cytokines, growth factors and metabolic signaling pathways to enable successful liver regeneration. One intracellular mediator that acts as a signal integration node for these processes is the serine-threonine kinase Akt/PKB (Akt). To investigate the contribution of Akt during hepatic regeneration, we performed partial hepatectomy in mice lacking Akt1, Akt2 or both isoforms. We found that absence of Akt1 or Akt2 does not influence liver regeneration after partial hepatectomy. However, hepatic-specific Akt1 and Akt2 null mice show impaired liver regeneration and increase mortality. The major abnormal cellular events observed in total Akt deficient livers were a marked reduction in cell proliferation, cell hypertrophy, glycogenesis and lipid droplets formation. Most importantly, liver-specific deletion of FoxO1, a transcription factor regulated by Akt, rescued the hepatic regenerative capability in Akt1 and Akt2 deficient mice and normalized the cellular events associated with liver regeneration. These results establish an essential role for the Akt-FoxO1 signaling pathway during liver regeneration that has not been previously described.

Rationale Recently, there has been significant interest in the therapeutic administration of miRNA mimics and inhibitors to treat cardiovascular disease. In particular, miR-27b has emerged as a regulatory hub in cholesterol and lipid metabolism and potential therapeutic target for treating atherosclerosis. Despite this, the impact of miR-27b on lipid levels in vivo remains to be determined. As such, here we set out to further characterize the role of miR-27b in regulating cholesterol metabolism in vitro and to determine the effect of miR-27b overexpression and inhibition on circulating and hepatic lipids in mice. Methods and Results Our results identify miR-27b as an important regulator of LDLR activity in human and mouse hepatic cells through direct targeting of LDLR and LDLRAP1. In addition, we report that modulation of miR-27b expression affects ABCA1 protein levels and cellular cholesterol efflux to ApoA1 in human hepatic Huh7 cells. Overexpression of pre-miR-27b in the livers of wild-type mice using AAV8 vectors increased pre-miR-27b levels 50–fold and reduced hepatic ABCA1 and LDLR expression by 50% and 20%, respectively, without changing circulating and hepatic cholesterol and triglycerides. To determine the effect of endogenous miR-27b on circulating lipids, wild-type mice were fed a Western diet for one month and injected with 5 mg/kg of LNA control or LNA anti-miR-27b oligonucleotides. Following two weeks of treatment, the expression of ABCA1 and LDLR were increased by 10–20% in the liver, demonstrating effective inhibition of miR-27b function. Intriguingly, no differences in circulating and hepatic lipids were observed between treatment groups. Conclusions The results presented here provide evidence that short-term modulation of miR-27b expression in wild-type mice regulates hepatic LDLR and ABCA1 expression but does not influence plasma and hepatic lipid levels.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with familial autosomal dominant hypercholesterolemia and is a natural inhibitor of the LDL receptor (LDLr). PCSK9 is degraded by other proprotein convertases: PC5/6A and furin. Both PCSK9 and the LDLr are up-regulated by the hypocholesterolemic statins. Thus, inhibitors or repressors of PCSK9 should amplify their beneficial effects. In the present study, we showed that PPAR␣ activation counteracts PCSK9 induction by statins by repressing PCSK9 promoter activity and by increasing PC5/6A and furin expression. Quantification of mRNA and protein levels showed that various fibrates decreased PCSK9 and increased PC5/6A and furin expression. Fenofibric acid (FA) reduced PCSK9 protein content in immortalized human hepatocytes (IHH) as well as its cellular secretion. FA suppressed PCSK9 induction by statins or by the liver X receptor agonist TO901317. PCSK9 repression is occurring at the promoter level. We showed that PC5/6A and furin fibrate-mediated up-regulation is PPAR␣-dependent. As a functional test, we observed that FA increased by 30% the effect of pravastatin on the LDLr activity in vitro. In conclusion, fibrates simultaneously decreased PCSK9 expression while increasing PC5/6A and furin expression, indicating a broad action of PPAR␣ activation in proprotein convertase-mediated lipid homeostasis. Moreover, this study validates the functional relevance of a combined therapy associating PCSK9 repressors and statins.Proprotein convertase subtilisin/kexin type 9 (PCSK9) 3 is associated with autosomal dominant hypercholesterolemia (1) and is a natural inhibitor of the LDL receptor (LDLr) (2). Its autocatalytic activity is necessary to its processing and for the mature form to reach the LDLr and induce its degradation (3). This catalytic activity is not involved in LDLr degradation per se. Indeed, cellular or secreted mature PCSK9 probably acts as a chaperone and prevents the LDLr from being recycled by binding to its EGFA domain (4 -10). Accordingly, hepatic transient overexpression of wild-type PCSK9 in mice (11)(12)(13)(14) or elevated circulating levels in parabiotic mice (15) decrease the LDLr expression and lead to hypercholesterolemia. It has been shown that the proprotein convertase furin and to a lesser extent PC5/6A cleave PCSK9 into a secreted, truncated, inactive form (16). Interestingly, some "gain of function" mutations do not necessarily induce a higher affinity of PCSK9 for the LDLr, but decrease PCSK9 sensitivity to this degradation (4). In contrast, PCSK9 deficiency lowers plasmatic cholesterol concentration and confers protection against cardiovascular disease (17-20). Thus, treatments inhibiting PCSK9 synthesis, processing, or binding to the LDLr could be useful for hypercholesterolemic patients who do not reach therapeutic goals. In particular, these inhibitors could be added to statins to amplify their effect on the LDLr activity. Surprisingly, statins also increase PCSK9 expression via SREBP-2, a pathway that exerts a break on ...

BackgroundPCSK9 (Proprotein Convertase Subtilisin Kexin type 9) is a circulating protein that promotes hypercholesterolemia by decreasing hepatic LDL receptor protein. Under non interventional conditions, its expression is driven by sterol response element binding protein 2 (SREBP2) and follows a diurnal rhythm synchronous with cholesterol synthesis. Plasma PCSK9 is associated to LDL-C and to a lesser extent plasma triglycerides and insulin resistance. We aimed to verify the effect on plasma PCSK9 concentrations of dietary interventions that affect these parameters.MethodsWe performed nutritional interventions in young healthy male volunteers and offspring of type 2 diabetic (OffT2D) patients that are more prone to develop insulin resistance, including: i) acute post-prandial hyperlipidemic challenge (n=10), ii) 4 days of high-fat (HF) or high-fat/high-protein (HFHP) (n=10), iii) 7 (HFruc1, n=16) or 6 (HFruc2, n=9) days of hypercaloric high-fructose diets. An acute oral fat load was also performed in two patients bearing the R104C-V114A loss-of-function (LOF) PCSK9 mutation. Plasma PCSK9 concentrations were measured by ELISA. For the HFruc1 study, intrahepatocellular (IHCL) and intramyocellular lipids were measured by 1H magnetic resonance spectroscopy. Hepatic and whole-body insulin sensitivity was assessed with a two-step hyperinsulinemic-euglycemic clamp (0.3 and 1.0 mU.kg-1.min-1).FindingsHF and HFHP short-term diets, as well as an acute hyperlipidemic oral load, did not significantly change PCSK9 concentrations. In addition, post-prandial plasma triglyceride excursion was not altered in two carriers of PCSK9 LOF mutation compared with non carriers. In contrast, hypercaloric 7-day HFruc1 diet increased plasma PCSK9 concentrations by 28% (p=0.05) in healthy volunteers and by 34% (p=0.001) in OffT2D patients. In another independent study, 6-day HFruc2 diet increased plasma PCSK9 levels by 93% (p<0.0001) in young healthy male volunteers. Spearman’s correlations revealed that plasma PCSK9 concentrations upon 7-day HFruc1 diet were positively associated with plasma triglycerides (r=0.54, p=0.01) and IHCL (r=0.56, p=0.001), and inversely correlated with hepatic (r=0.54, p=0.014) and whole-body (r=−0.59, p=0.0065) insulin sensitivity.ConclusionsPlasma PCSK9 concentrations vary minimally in response to a short term high-fat diet and they are not accompanied with changes in cholesterolemia upon high-fructose diet. Short-term high-fructose intake increased plasma PCSK9 levels, independent on cholesterol synthesis, suggesting a regulation independent of SREBP-2. Upon this diet, PCSK9 is associated with insulin resistance, hepatic steatosis and plasma triglycerides.