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Background and aims Familial hypercholesterolemia (FH) is a monogenic disease characterized by high levels of low-density lipoprotein cholesterol and premature atherosclerotic cardiovascular disease. FH is caused by loss of function mutations in genes encoding LDL receptor (LDLR), and Apolipoprotein B (APOB) or gain of function (GOF) mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9). In this study, we identified a novel variant in PCSK9, p.(Arg499His), located in the C-terminal domain, in two unrelated FH patients from Spain and Italy. Methods We studied familial segregation and determined variant activity in vitro. Results We determined PCSK9 expression, secretion and activity of the variant in transfected HEK293 cells; extracellular activity of the recombinant p.(Arg499His) PCSK9 variant in HEK 293 and HepG2 cells; PCSK9 affinity to the LDL receptor at neutral and acidic pH; the mechanism of action of the p.(Arg499His) PCSK9 variant by co-transfection with a soluble construct of the LDL receptor and by determining total PCSK9 intracellular accumulation when endosomal acidification is impaired and when an excess of soluble LDLr is present in the culture medium. Our results show high LDL-C concentrations and FH phenotype in p.(Arg499His) carriers. In vitro functional characterization shows that p.(Arg499His) PCSK9 variant causes a reduction in LDLr expression and LDL uptake. An intracellular activity for this variant is also shown when blocking the activity of secreted PCSK9 and by inhibiting endosomal acidification. Conclusions We demonstrated that p.(Arg499His) PCSK9 variant causes a direct intracellular degradation of LDLr therefore causing FH by reducing LDLr availability.

Familial hypercholesterolaemia (FH) is an autosomal disorder of lipid metabolism presenting with increased cardiovascular risk. LDLR mutations are the cause of disease in 90% of the cases but functional studies have only been performed for about 15% of all LDLR variants. In the Portuguese Familial Hypercholesterolemia Study (PFHS), 142 unique LDLR alterations were identified and 44 (30%) lack functional characterization. The aim of the present work is to increase evidence for variant classification by performing functional characterization of 13 LDLR missense alterations found in Portugal and in 20 other countries., Funding was obtained from UID/MULTI/04046/2019 Research Unit grant from FCT, Portugal (to BioISI) and for the Basque Government (Grupos Consolidados IT-1264-19). UGG was supported by Fundación Biofísica Bizkaia. ABV. was supported by Programa de especialización de Personal Investigador Doctor en la UPV/EHU (2019) 2019–2020.

Insulin resistance (IR) is one of the key contributing factors in the development of type 2 diabetes mellitus (T2DM). However, the molecular mechanisms leading to IR are still unclear. The implication of microRNAs (miRNAs) in the pathophysiology of multiple cardiometabolic pathologies, including obesity, atherosclerotic heart failure and IR, has emerged as a major focus of interest in recent years. Indeed, upregulation of several miRNAs has been associated with obesity and IR. Among them, miR-27b is overexpressed in the liver in patients with obesity, but its role in IR has not yet been thoroughly explored. In this study, we investigated the role of miR-27b in regulating insulin signaling in hepatocytes, both in vitro and in vivo. Therefore, assessment of the impact of miR-27b on insulin resistance through the hepatic tissue is of special importance due to the high expression of miR-27b in the liver together with its known role in regulating lipid metabolism. Notably, we found that miR-27b controls post-transcriptional expression of numerous components of the insulin signaling pathway including the insulin receptor (INSR) and insulin receptor substrate 1 (IRS1) in human hepatoma cells. These results were further confirmed in vivo showing that overexpression and inhibition of hepatic miR-27 enhances and suppresses hepatic INSR expression and insulin sensitivity, respectively. This study identified a novel role for miR-27 in regulating insulin signaling, and this finding suggests that elevated miR-27 levels may contribute to early development of hepatic insulin resistance. This work was supported by the Basque Government (Grupos Consolidados IT-1264-19). A.B.-V. was supported by Programa de especialización de Personal Investigador Doctor en la UPV/EHU (2019) 2019-2020. U.G-G. was supported by Fundación Biofísica Bizkaia. S.J. was supported by a grant PIF (2017–2018), Gobierno Vasco. We sincerely thank Haziq Siddiqi (Harvard Medical School) for his critical reading and editing of this manuscript.

The primary genetic cause of familial hypercholesterolemia (FH) is related to mutations in the LDLR gene encoding the Low-density Lipoprotein Receptor. LDLR structure is organized in 5 different domains, including an EGF-precursor homology domain that plays a pivotal role in lipoprotein release and receptor recycling. Mutations in this domain constitute 51.7% of the total missense variants described in LDLR. The aim of the present work was to analyse how clinically significant variants in the EGF-precursor homology domain impact LDLR. The activity of sixteen LDLR variants was functionally characterized by determining LDLR expression by Western blot and LDLR expression, LDL binding capacity and uptake, and LDLR recycling activity by flow cytometry in transfected CHO-ldlA7 cells. Of the analysed variants, we found six non-pathogenic LDLR variants and ten pathogenic variants distributed as follow: three class 3 variants; four class 2 variants; and three class 5 variants. These results can be incorporated into clinical management of patients by helping guide the appropriate level of treatment intensity depending on the extent of loss of LDLR activity. This data can also contribute to cascade-screening for pathogenic FH variants. We thank Prof. Monty Krieger for kindly providing CHO-ldlA7 cells. We sincerely thank Haziq Siddiqi (Harvard Medical School) for his critical reading and editing of this manuscript and Professor Katrine T. Schjoldager (Copenhagen Center for Glycomics) for her invaluable research assistance on glycosylation analysis of p.(Ser584Pro) LDLR variant. This work was supported by the Basque Government (Grupos Consolidados IT-1264-19). A.B.-V. was supported by a grant PIF (2014–2015), Gobierno Vasco and DOKBERRI 2019-I. S.J. was supported by a grant PIF (2017–2018), Gobierno Vasco. U.G-G. and R.A-E. were supported by Fundación Biofísica Bizkaia

Type 2 Diabetes Mellitus (T2DM), one of the most common metabolic disorders, is caused by a combination of two primary factors: defective insulin secretion by pancreatic β-cells and the inability of insulin-sensitive tissues to respond appropriately to insulin. Because insulin release and activity are essential processes for glucose homeostasis, the molecular mechanisms involved in the synthesis and release of insulin, as well as in its detection are tightly regulated. Defects in any of the mechanisms involved in these processes can lead to a metabolic imbalance responsible for the development of the disease. This review analyzes the key aspects of T2DM, as well as the molecular mechanisms and pathways implicated in insulin metabolism leading to T2DM and insulin resistance. For that purpose, we summarize the data gathered up until now, focusing especially on insulin synthesis, insulin release, insulin sensing and on the downstream effects on individual insulin-sensitive organs. The review also covers the pathological conditions perpetuating T2DM such as nutritional factors, physical activity, gut dysbiosis and metabolic memory. Additionally, because T2DM is associated with accelerated atherosclerosis development, we review here some of the molecular mechanisms that link T2DM and insulin resistance (IR) as well as cardiovascular risk as one of the most important complications in T2DM. This work was supported by the Basque Government (Grupos Consolidados IT-1264-19). U.G-G. was supported by Fundación Biofísica Bizkaia. A.B.-V. was supported by Programa de especializaci.n de Personal Investigador Doctor en la UPV/EHU (2019) 2019–2020. S.J. and A.L-S. were supported by a grant PIF (2017–2018) and (2019–2020), Gobierno Vasco, respectively. A.L.-S. was partially supported by Fundación Biofísica Bizkaia.

Cardiovascular disease (CVD), the leading cause of mortality worldwide is primarily caused by atherosclerosis, which is promoted by the accumulation of low-density lipoproteins into the intima of large arteries. Multiple nanoparticles mimicking natural HDL (rHDL) have been designed to remove cholesterol excess in CVD therapy. The goal of this investigation was to assess the cholesterol efflux efficiency of rHDLs with different lipid compositions, mimicking different maturation stages of high-density lipoproteins (HDLs) occurring in vivo. Methods: the cholesterol efflux activity of soybean PC (Soy-PC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), DPPC:Chol:1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (LysoPC) and DPPC:18:2 cholesteryl ester (CE):LysoPC rHDLs was determined in several cell models to investigate the contribution of lipid composition to the effectiveness of cholesterol removal. Results: DPPC rHDLs are the most efficient particles, inducing cholesterol efflux in all cellular models and in all conditions the effect was potentiated when the ABCA1 transporter was upregulated. Conclusions: DPPC rHDLs, which resemble nascent HDL, are the most effective particles in inducing cholesterol efflux due to the higher physical binding affinity of cholesterol to the saturated long-chain-length phospholipids and the favored cholesterol transfer from a highly positively curved bilayer, to an accepting planar bilayer such as DPPC rHDLs. The physicochemical characteristics of rHDLs should be taken into consideration to design more efficient nanoparticles to promote cholesterol efflux. This work was supported by the Basque Government (Grupos Consolidados IT-1264-19). U.G.-G. was supported by Fundación Biofísica Bizkaia. A.B.-V. was supported by Programa de especialización de Personal Investigador Doctor en la UPV/EHU (2019) 2019–2020. S.J.-B. and A.L.-S. were supported by a grant PIF (2017–2018) and (2019–2020), Gobierno Vasco, respectively. A.L.-S. was partially supported by Fundación Biofísica Bizkaia.

Statins are the gold-standard treatment for the prevention of primary and secondary cardiovascular disease, which is the leading cause of mortality worldwide. Despite the safety and relative tolerability of statins, observational studies, clinical trials and meta-analyses indicate an increased risk of developing new-onset type 2 diabetes mellitus (T2DM) after long-term statin treatment. It has been shown that statins can impair insulin sensitivity and secretion by pancreatic β-cells and increase insulin resistance in peripheral tissues. The mechanisms involved in these processes include, among others, impaired Ca2+ signaling in pancreatic β-cells, down-regulation of GLUT-4 in adipocytes and compromised insulin signaling. In addition, it has also been described that statins’ impact on epigenetics may also contribute to statin-induced T2DM via differential expression of microRNAs. This review focuses on the evidence and mechanisms by which statin therapy is associated with the development of T2DM. This review describes the multifactorial combination of effects that most likely contributes to the diabetogenic effects of statins. Clinically, these findings should encourage clinicians to consider diabetes monitoring in patients receiving statin therapy in order to ensure early diagnosis and appropriate management. This work was supported by the Basque Government (Grupos Consolidados IT-1264-19). U.G.-G. was supported by Fundación Biofísica Bizkaia. A.B.-V. was supported by Programa de especialización de Personal Investigador Doctor en la UPV/EHU (2019) 2019–2020. S.J. and A.L.-S. were supported by a grant PIF (2017–2018) and (2019–2020), Gobierno Vasco, respectively. A.L.-S. was partially supported by Fundación Biofísica Bizkaia.

Cholesterol is an essential component of cell barrier formation and signaling transduction involved in many essential physiologic processes. For this reason, cholesterol metabolism must be tightly controlled. Cell cholesterol is mainly acquired from two sources: Dietary cholesterol, which is absorbed in the intestine and, intracellularly synthesized cholesterol that is mainly synthesized in the liver. Once acquired, both are delivered to peripheral tissues in a lipoprotein dependent mechanism. Malfunctioning of cholesterol metabolism is caused by multiple hereditary diseases, including Familial Hypercholesterolemia, Sitosterolemia Type C and Niemann-Pick Type C1. Of these, familial hypercholesterolemia (FH) is a common inherited autosomal co-dominant disorder characterized by high plasma cholesterol levels. Its frequency is estimated to be 1:200 and, if untreated, increases the risk of premature cardiovascular disease. This review aims to summarize the current knowledge on cholesterol metabolism and the relation of FH to cholesterol homeostasis with special focus on the genetics, diagnosis and treatment. This work was supported by ELKARTEK 2016 and the Basque Government (Grupos Consolidados IT849-13). A.B.-V. and S.J. were supported by a grant PIF (2014-2015) and (2018-2021), Gobierno Vasco respectively.

Familial Hypercholesterolemia (FH) is a common genetic disorder caused most often by mutations in the Low Density Lipoprotein Receptor gene (LDLr) leading to high blood cholesterol levels, and ultimately to development of premature coronary heart disease. Genetic analysis and subsequent cascade screening in relatives allow diagnosis of FH at early stage, especially relevant to diagnose children. So far, more than 2300 LDLr variants have been described but only a minority of them have been functionally analysed to evaluate their pathogenicity in FH. Thus, identifying pathogenic mutations in LDLr is a long-standing challenge in the field. In this study, we investigated in vitro the activity p.(Asp47Asn) and p.(Thr62Met) LDLr variants, both in the LR1 region. We used CHO-ldlA7 transfected cells with plasmids carrying p.(Asp47Asn) or p.(Thr62Met) LDLr variants to analyse LDLr expression by FACS and immunoblotting, LDL binding and uptake was determined by FACS and analysis of mutation effects was assessed in silico. The in vitro activity assessment of p.(Asp47Asn) and p.(Thr62Met) LDLr variants shows a fully functional LDL binding and uptake activities. Therefore indicating that the three of them are non-pathogenic LDLr variants. These findings also emphasize the importance of in vitro functional LDLr activity studies to optimize the genetic diagnosis of FH avoiding the report of non-pathogenic variants and possible misdiagnose in relatives if cascade screening is carried out.

Background and aims Pathogenic mutations in the Low Density Lipoprotein Receptor gene (LDLR) cause Familial Hypercholesterolemia (FH), one of the most common genetic disorders with a prevalence as high as 1 in 200 in some populations. FH is an autosomal dominant disorder of lipoprotein metabolism characterized by high blood cholesterol levels, deposits of cholesterol in peripheral tissues such as tendon xanthomas and accelerated atherosclerosis. To date, 2500 LDLRvariants have been identified in the LDLR gene; however, only a minority of them have been experimentally characterized and proven to be pathogenic. Here we investigated the role of Cys46 located in the first repeat of the LDL receptor binding domain in recognition of apolipoproteins. Methods Activity of the p.(Cys46Gly) LDLR variant was assessed by immunoblotting and flow cytometry in CHO-/d/A7 expressing the receptor variant. Affinity of p.(Cys46Gly) for LDL and VLDL was determined by solid-phase immunoassays and in silico analysis was used to predict mutation effects. Results and conclusion Functional characterization of p.(Cys46Gly) LDLRvariant showed impaired LDL and VLDL binding and uptake activity. Consistent with this, solid-phase immunoassays showed the p. (Cys46Gly) LDLR variant has decreased binding affinity for apolipoproteins. These results indicate the important role of Cys46 in LDL receptor activity and highlight the role of LR1 in LDLr activity modulation. This study reinforces the significance of in vitro functional characterization of LDL receptor activity in developing an accurate approach to FH genetic diagnosis. This is of particular importance because it enables clinicians to tailor personalized treatments for patients' mutation profile.

Familial hypercholesterolemia (FH) is an autosomal dominant disorder characterized by high blood-cholesterol levels mostly caused by mutations in the low-density lipoprotein receptor (LDLr). With a prevalence as high as 1/200 in some populations, genetic screening for pathogenic LDLr mutations is a cost-effective approach in families classified as definite' or probable' FH and can help to early diagnosis. However, with over 2000 LDLr variants identified, distinguishing pathogenic mutations from benign mutations is a long-standing challenge in the field. In 1998, the World Health Organization (WHO) highlighted the importance of improving the diagnosis and prognosis of FH patients thus, identifying LDLr pathogenic variants is a longstanding challenge to provide an accurate genetic diagnosis and personalized treatments. In recent years, accessible methodologies have been developed to assess LDLr activity in vitro, providing experimental reproducibility between laboratories all over the world that ensures rigorous analysis of all functional studies. In this review we present a broad spectrum of functionally characterized missense LDLr variants identified in patients with FH, which is mandatory for a definite diagnosis of FH. This work was supported by ELKARTEK 2016 and and the Basque Government (Grupos Consolidados IT849-13). A.B.-V. and S.J. were supported by a grant PIF (2014-2015) and (2018-2021), Gobierno Vasco respectively.