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Genetic variation at rs4240624 on chromosome 8 is associated with an attenuated signal on hepatic computerized tomography, which has been attributed to changes in hepatic fat. The closest coding gene to rs4240624, PPP1R3B, encodes a protein that promotes hepatic glycogen synthesis. Here, we performed studies to determine whether the x-ray attenuation associated with rs4240624 is due to differences in hepatic glycogen or hepatic triglyceride content (HTGC). A sequence variant in complete linkage disequilibrium with rs4240624, rs4841132, was genotyped in the Dallas Heart Study (DHS), the Dallas Liver Study, and the Copenhagen Cohort (n = 112,428) of whom 1,539 had nonviral liver disease. The minor A-allele of rs4841132 was associated with increased hepatic x-ray attenuation (n = 1,572; P = 4 × 10-5 ), but not with HTGC (n = 2,674; P = 0.58). Rs4841132-A was associated with modest, but significant, elevations in serum alanine aminotransferase (ALT) in the Copenhagen Cohort (P = 3 × 10-4 ) and the DHS (P = 0.004), and with odds ratios for liver disease of 1.13 (95% CI, 0.97-1.31) and 1.23 (1.01-1.51), respectively. Mice lacking protein phosphatase 1 regulatory subunit 3B (PPP1R3B) were deficient in hepatic glycogen, whereas HTGC was unchanged. Hepatic overexpression of PPP1R3B caused accumulation of hepatic glycogen and elevated plasma levels of ALT, but did not change HTGC.ConclusionThese observations are consistent with the notion that the minor allele of rs4841132 promotes a mild form of hepatic glycogenosis that is associated with hepatic injury. (Hepatology 2018;67:2182-2195).

A missense variant in patatin-like phospholipase domain-containing protein 3 [PNPLA3(I148M)] is the most impactful genetic risk factor for fatty liver disease (FLD). We previously showed that PNPLA3 is ubiquitylated and subsequently degraded by proteasomes and autophagosomes and that the PNPLA3(148M) variant interferes with this process. To define the machinery responsible for PNPLA3 turnover, we used small interfering (si)RNAs to inactivate components of the ubiquitin proteasome system. Inactivation of bifunctional apoptosis regulator (BFAR), a membrane-bound E3 ubiquitin ligase, reproducibly increased PNPLA3 levels in two lines of cultured hepatocytes. Conversely, overexpression of BFAR decreased levels of endogenous PNPLA3 in HuH7 cells. BFAR and PNPLA3 co-immunoprecipitated when co-expressed in cells. BFAR promoted ubiquitylation of PNPLA3 in vitro in a reconstitution assay using purified, epitope-tagged recombinant proteins. To confirm that BFAR targets PNPLA3, we inactivated Bfar in mice. Levels of PNPLA3 protein were increased twofold in hepatic lipid droplets of Bfar−/− mice with no associated increase in PNPLA3 mRNA levels. Taken together these data are consistent with a model in which BFAR plays a role in the post-translational degradation of PNPLA3. The identification of BFAR provides a potential target to enhance PNPLA3 turnover and prevent FLD. [ABSTRACT FROM AUTHOR]

The X-ray structure of human ABCG5/ABCG8 heterodimer in a nucleotide-free state, being the first atomic model of an ABC sterol transporter. Human ABCG5/ABCG8 sterol transporter Cholesterol is an essential component of vertebrate cell membranes. Animals maintain sterol balance by limiting dietary sterol uptake from the gut and promoting sterol secretion from hepatocytes into bile. These physiological processes are mediated by ABCG5/ABCG8, a heterodimeric ABC transporter. These authors have solved the X-ray crystal structure of the human ABCG5/ABCG8 heterodimer in a nucleotide-free state. As well as being the first atomic model of an ABC sterol transporter, the structure provides mechanistic insights into sterol transport and establishes a framework for understanding mutations responsible for sitosterolaemia, a human disease characterized by premature atherosclerosis. ATP binding cassette (ABC) transporters play critical roles in maintaining sterol balance in higher eukaryotes. The ABCG5/ABCG8 heterodimer (G5G8) mediates excretion of neutral sterols in liver and intestines.sup.1,2,3,4,5. Mutations disrupting G5G8 cause sitosterolaemia, a disorder characterized by sterol accumulation and premature atherosclerosis. Here we use crystallization in lipid bilayers to determine the X-ray structure of human G5G8 in a nucleotide-free state at 3.9 Å resolution, generating the first atomic model of an ABC sterol transporter. The structure reveals a new transmembrane fold that is present in a large and functionally diverse superfamily of ABC transporters. The transmembrane domains are coupled to the nucleotide-binding sites by networks of interactions that differ between the active and inactive ATPases, reflecting the catalytic asymmetry of the transporter. The G5G8 structure provides a mechanistic framework for understanding sterol transport and the disruptive effects of mutations causing sitosterolaemia., Author(s): Jyh-Yeuan Lee [sup.1] , Lisa N. Kinch [sup.2] [sup.3] [sup.4] , Dominika M. Borek [sup.2] [sup.3] , Jin Wang [sup.1] , Junmei Wang [sup.5] , Ina L. Urbatsch [sup.6] [...]

Background and aims: Susceptibility to fatty liver disease (FLD) varies among individuals and between racial/ethnic groups. Several genetic variants influence FLD risk, but whether these variants explain racial/ethnic differences in FLD prevalence is unclear. We examined the contribution of genetic risk factors to racial/ethnic‐specific differences in FLD. Methods: A case–control study comparing FLD patients (n = 1194) and population‐based controls (n = 3120) was performed. Patient characteristics, FLD risk variants (PNPLA3‐rs738409 + rs6006460, TM6SF2‐rs58542926, HSD17B13‐rs80182459 + rs72613567, MBOAT7/TMC4‐rs641738, and GCKR‐rs1260326) and a multi‐locus genetic risk score (GRS) were examined. The odds of FLD for individuals with different risk factor burdens were determined. Results: Hispanics and Whites were over‐represented (56% vs. 38% and 36% vs. 29% respectively) and Blacks under‐represented (5% vs. 23%) among FLD patients, compared to the population from which controls were selected (p <.001). Among cases and controls, Blacks had a lower and Hispanics a greater, net number of risk alleles than Whites (p <.001). GRS was associated with increased odds of FLD (ORQ5vsQ1 = 8.72 [95% CI = 5.97–13.0], p = 9.8 × 10−28), with the association being stronger in Hispanics (ORQ5vsQ1 = 14.8 [8.3–27.1]) than Blacks (ORQ5vsQ1 = 3.7 [1.5–11.5], P‐interaction = 0.002). After accounting for GRS, the odds of FLD between Hispanics and Whites did not differ significantly (OR = 1.06 [0.87–1.28], p =.58), whereas Blacks retained much lower odds of FLD (OR = 0.21, [0.15–0.30], p <.001). Conclusions: Blacks had a lower and Hispanics a greater FLD risk allele burden than Whites. These differences contributed to, but did not fully explain, racial/ethnic differences in FLD prevalence. Identification of additional factors protecting Blacks from FLD may provide new targets for prevention and treatment of FLD. [ABSTRACT FROM AUTHOR]

A genetic variant in PNPLA3 [(PNPLA3.sup.I148M]), a triacylglycerol (TAG) hydrolase, is a major risk factor for non-alcoholic fatty liver disease (NAFLD); however, the mechanism underlying this association is not known. To develop an animal model of PNPLA3-induced fatty liver disease, we generated transgenic mice that overexpress similar amounts of wild-type PNPLA3 [(PNPLA3.sup.WT]) or mutant PNPLA3 [(PNPLA3.sup.I148M]) either in liver or adipose tissue. Overexpression of the transgenes in adipose tissue did not affect liver fat content. Expression of [PNPLA3.sup.I148M], but not [PNPLA3.sup.WT], in liver recapitulated the fatty liver phenotype as well as other metabolic features associated with this allele in humans. Metabolic studies provided evidence for 3 distinct alterations in hepatic TAG metabolism in [PNPLA3.sup.I148M] transgenic mice: increased formation of fatty acids and TAG, impaired hydrolysis of TAG, and relative depletion of TAG long-chain polyunsaturated fatty acids. These findings suggest that PNPLA3 plays a role in remodeling TAG in lipid droplets, as they accumulate in response to food intake, and that the increase in hepatic TAG levels associated with the I148M substitution results from multiple changes in hepatic TAG metabolism. The development of an animal model that recapitulates the metabolic phenotype of the allele in humans provides a new platform in which to elucidate the role of [PNLPA3.sup.I148M] in NAFLD., Introduction Higher organisms stockpile triacylglycerol (TAG) in adipose tissue as a buffer against fluctuations in food availability and energy demands. TAG is usually maintained at much lower concentrations in other [...]

All protein-coding regions of the genome (the 'exome') are sequenced in two family members with combined hypolipidemia, marked by extremely low plasma levels of low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol and triglycerides. The analysis of ANGPTL3 (the angiopoietinlike 3 protein) mutations has highlighted a role for the gene in LDL cholesterol metabolism in humans and has displayed the usefulness of exome sequencing for identification of novel genetic causes of inherited disorders.

The relative activity of lipoprotein lipase (LPL) in different tissues controls the partitioning of lipoproteinderived fatty acids between sites of fat storage (adipose tissue) and oxidation (heart and skeletal muscle). Here we used a reverse genetic strategy to test the hypothesis that 4 angiopoietin-like proteins (ANGPTL3, -4, -5, and -6) play key roles in triglyceride (TG) metabolism in humans. We re-sequenced the coding regions of the genes encoding these proteins and identified multiple rare nonsynonymous (NS) sequence variations that were associated with low plasma TG levels but not with other metabolic phenotypes. Functional studies revealed that all mutant alleles of ANGPTL3 and ANGPTL4 that were associated with low plasma TG levels interfered either with the synthesis or secretion of the protein or with the ability of the ANGPTL protein to inhibit LPL. A total of 1% of the Dallas Heart Study population and 4% of those participants with a plasma TG in the lowest quartile had a rare loss-of-function mutation in ANGPTL3, ANGPTL4, or ANGPTL5. Thus, ANGPTL3, ANGPTL4, and ANGPTL5, but not ANGPTL6, play nonredundant roles in TG metabolism, and multiple alleles at these loci cumulatively contribute to variability in plasma TG levels in humans., Introduction In individuals consuming Western diets, more than 100 grams of triglycerides (TGs) are transported each day from the liver and small intestines to peripheral tissues. The partitioning of TG [...]

Proprotein convertase subtilisin-like kexin type 9 (PCSK9) is a newly discovered serine protease that destroys low density lipoprotein (LDL) receptors in liver and thereby controls the level of LDL in plasma. Mutations that increase PCSK9 activity cause hypercholesterolemia and coronary heart disease (CHD), mutations that inactivate PCSK9 have the opposite effect, lowering LDL levels and reducing CHD. Although the mechanism of PCSK9 action is not yet clear, the protease provides a new therapeutic target to lower plasma levels of LDL and prevent CHD.

Genetic defects in LDL clearance result in severe hypercholesterolemia and premature atherosclerosis. Mutations in the LDL receptor (LDLR) cause familial hypercholesterolemia (FH), the most severe form of genetic hypercholesterolemia. A [...]

The effect of loss-of-function mutations on the synthesis and secretion of proprotein convertase subtilisin/kexin type 9 (PCSK9) was examined. It predicted that circulating levels of PCSK9 was lower in individuals with loss-of-function mutations and serine protease is present in the circulation and identified the first known compound heterozygote with two inactivating mutations in PCSK9.

The incidence of coronary heart disease was compared over a 15-year interval in a study according to the presence or absence of sequence variants in the proprotein convertase substilisin/kexin type 9 serine protease gene (PCSK9) that are associated with reduced plasma levels of low density lipoprotein (LDL) cholesterol. The data indicate that moderate lifelong reduction in the plasma level of LDL cholesterol is associated with a substantial reduction in the incidence of coronary events even in populations with a high prevalence of non-lipid-related cardiovascular factors.

The structural features of sterols required to support mammalian cell growth have not been fully defined. Here, we use mutant CHO cells that synthesize only small amounts of cholesterol to test the capacity of various sterols to support growth. Sterols with minor modifications of the side chain (e.g., campesterol, [beta]-sitosterol, and desmosterol) supported long-term growth of mutant cells, but sterols with more complex modifications of the side chain, the sterol nucleus, or the 3-hydroxy group did not. After 60 days in culture, the exogenous sterol comprised >90% of cellular sterols. Inactivation of residual endogenous synthesis with the squalene epoxidase inhibitor NB-598 prevented growth in [beta]-sitosterol and greatly reduced growth in campesterol. Growth of ceils cultured in [beta]-sitosterol and NB-598 was restored by adding small amounts of cholesterol to the medium. Surprisingly, enantiomeric cholesterol also supported cell growth, even in the presence of NB-598. Thus, sterols fulfill two roles in mammalian cells: (i) a bulk membrane requirement in which phytosterols can substitute for cholesterol and (ii) other processes that specifically require small amounts of cholesterol but are not enantioselective. ent-cholesterol | phytosterols | NB-598

Despite the increasing prevalence of nonalcoholic fatty liver disease (NAFLD), the criteria used to diagnose the disorder remain poorly defined. Localized proton magnetic resonance spectroscopy (MRS) accurately measures hepatic triglyceride content (HTGC) but has been used only in small research studies. Here, MRS was used to analyze the distribution of HTGC in 2,349 participants from the Dallas Heart Study (DHS). The reproducibility of the procedure was validated by showing that duplicate HTGC measurements were high correlated (r = 0.99, P < 0.001) and that the coefficient of variation between measurements was low (8.5%). Intake of a high-fat meal did not significantly affect the measurements, and values were similar when measurements were made from the right and left hepatic lobes. To determine the 'upper limit of normal' for HTGC, the distribution of HTGC was examined in the 345 subjects from the DHS who had no identifiable risk factors for hepatic steatosis (nonobese, nondiabetic subjects with minimal alcohol consumption, normal liver function tests, and no known liver disease). The 95th percentile of HTGC in these subjects was 5.56%, which corresponds to a hepatic triglyceride level of 55.6 mg/g. With this value as a cutoff, the prevalence of hepatic steatosis in Dallas County was estimated to be 33.6%. Thus MRS provides a sensitive, quantitative, noninvasive method to measure HTGC and, when applied to a large urban US population, revealed a strikingly high prevalence of hepatic steatosis. fatty liver

Atherosclerosis is the primary cause of cardiovascular disease, and the risk for atherosclerosis is inversely proportional to circulating levels of high-density lipoprotein (HDL) cholesterol. However, the mechanisms by which HDL is atheroprotective are complex and not well understood. Here we show that HDL stimulates endothelial nitric oxide synthase (eNOS) in cultured endothelial cells. In contrast, eNOS is not activated by purified forms of the major HDL apolipoproteins ApoA-I and ApoA-II or by low-density lipoprotein. Heterologous expression experiments in Chinese hamster ovary cells reveal that scavenger receptor-BI (SR-BI) mediates the effects of HDL on the enzyme. HDL activation of eNOS is demonstrable in isolated endothelial-cell caveolae where SR-BI and eNOS are colocalized, and the response in isolated plasma membranes is blocked by antibodies to ApoA-I and SR-BI, but not by antibody to ApoA-II. HDL also enhances endothelium- and nitric-oxide-dependent relaxation in aortae from wild-type mice, but not in aortae from homozygous null SR-BI knockout mice. Thus, HDL activates eNOS via SR-BI through a process that requires ApoA-I binding. The resulting increase in nitric-oxide production might be critical to the atheroprotective properties of HDL and ApoA-I., Author(s): Ivan S. Yuhanna [1]; Yan Zhu [4]; Blair E. Cox [1]; Lisa D. Hahner [1]; Sherri Osborne-Lawrence [1]; Ping Lu [4]; Yves L. Marcel [5]; Richard G.W. Anderson [3]; [...]