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Fatty liver disease (FLD) is a disorder in which accumulation of triglycerides (TGs) in the liver can lead to inflammation, fibrosis, and cirrhosis. Previously, we identified a variant (I148M) in patatin-like phospholipase domain-containing protein 3 (PNPLA3) that is strongly associated with FLD, but the mechanistic basis for the association remains elusive. Although PNPLA3 has TG hydrolase activity in vitro, inactivation or overexpression of the WT protein in mice does not cause steatosis. In contrast, expression of two catalytically defective forms of PNPLA3 (I148M or S47A) in sucrose-fed mice causes accumulation of both PNPLA3 and TGs on hepatic lipid droplets (LDs). To determine if amassing PNPLA3 on LDs is a cause or consequence of steatosis, we engineered a synthetic isoform of PNPLA3 that uncouples protein accumulation from loss of enzymatic activity. Expression of a ubiquitylation-resistant form of PNPLA3 in mice caused accumulation of PNPLA3 on hepatic LDs and development of FLD. Lowering PNPLA3 levels by either shRNA knockdown or proteolysis-targeting chimera (PROTAC)-mediated degradation reduced liver TG content in mice overexpressing PNPLA3(148M). Taken together, our results show that the steatosis associated with PNPLA3(148M) is caused by accumulation of PNPLA3 on LDs.

Nonalcoholic fatty liver disease (NAFLD) is a burgeoning health problem that affects one-third of adults and an increasing number of children in developed countries. The disease begins with the aberrant accumulation of triglyceride in the liver, which in some individuals elicits an inflammatory response that can progress to cirrhosis and liver cancer. Although NAFLD is strongly associated with obesity and insulin resistance, its pathogenesis remains poorly understood, and therapeutic options are limited. Here, we discuss recent mechanistic insights into NAFLD, focusing primarily on those that have emerged from human genetic and metabolic studies.

Jonathan Cohen, Helen Hobbs and colleagues show that adiposity significantly amplifies the effects of PNPLA3 , TM6SF2 , and GCKR sequence variants on nonalcoholic fatty liver disease. They find that synergy between adiposity and genotype influences the full spectrum of the disease, from steatosis to hepatic inflammation and cirrhosis. Complex traits arise from the interplay between genetic and environmental factors. The actions of these factors usually appear to be additive, and few compelling examples of gene–environment synergy have been documented. Here we show that adiposity significantly amplifies the effect of three sequence variants (encoding PNPLA3 p.I148M, TM6SF2 p.E167K, and GCKR p.P446L) associated with nonalcoholic fatty liver disease (NAFLD). Synergy between adiposity and genotype promoted the full spectrum of NAFLD, from steatosis to hepatic inflammation to cirrhosis. We found no evidence of strong interaction between adiposity and sequence variants influencing other adiposity-associated traits. These results indicate that adiposity augments genetic risk of NAFLD at multiple loci that confer susceptibility to hepatic steatosis through diverse metabolic mechanisms.

Helen Hobbs and colleagues report an association between coding variation in PNPLA3 and susceptibility to nonalcoholic fatty liver disease. The associated alleles vary in frequency among Hispanics, African Americans and European Americans and contribute to differences in disease prevalence among these ancestry groups. Nonalcoholic fatty liver disease (NAFLD) is a burgeoning health problem of unknown etiology that varies in prevalence among ancestry groups. To identify genetic variants contributing to differences in hepatic fat content, we carried out a genome-wide association scan of nonsynonymous sequence variations ( n = 9,229) in a population comprising Hispanic, African American and European American individuals. An allele in PNPLA3 (rs738409[G], encoding I148M) was strongly associated with increased hepatic fat levels ( P = 5.9 × 10 −10 ) and with hepatic inflammation ( P = 3.7 × 10 −4 ). The allele was most common in Hispanics, the group most susceptible to NAFLD; hepatic fat content was more than twofold higher in PNPLA3 rs738409[G] homozygotes than in noncarriers. Resequencing revealed another allele of PNPLA3 (rs6006460[T], encoding S453I) that was associated with lower hepatic fat content in African Americans, the group at lowest risk of NAFLD. Thus, variation in PNPLA3 contributes to ancestry-related and inter-individual differences in hepatic fat content and susceptibility to NAFLD.

Angiopoietin-like proteins (ANGPTLs) play major roles in the trafficking and metabolism of lipids. Inactivation of ANGPTL3 , a gene located in an intron of DOCK7 , results in very low levels of LDL-cholesterol (C), HDL-C and triglyceride (TAG). We identified another ANGPTL family member, ANGPTL8 , which is located in the corresponding intron of DOCK6 . A variant in this family member (rs2278426, R59W) was associated with lower plasma LDL-C and HDL-C levels in three populations. ANGPTL8 is expressed in liver and adipose tissue, and circulates in plasma of humans. Expression of ANGPTL8 was reduced by fasting and increased by refeeding in both mice and humans. To examine the functional relationship between the two ANGPTL family members, we expressed ANGPTL3 at physiological levels alone or together with ANGPTL8 in livers of mice. Plasma TAG level did not change in mice expressing ANGPTL3 alone, whereas coexpression with ANGPTL8 resulted in hypertriglyceridemia, despite a reduction in circulating ANGPTL3. ANGPTL8 coimmunoprecipitated with the N-terminal domain of ANGPTL3 in plasma of these mice. In cultured hepatocytes, ANGPTL8 expression increased the appearance of N-terminal ANGPTL3 in the medium, suggesting ANGPTL8 may activate ANGPTL3. Consistent with this scenario, expression of ANGPTL8 in Angptl3 ⁻/⁻ mice failed to promote hypertriglyceridemia. Thus, ANGPTL8 , a paralog of ANGPTL3 that arose through duplication of an ancestral DOCK gene, regulates postprandial TAG and fatty acid metabolism by controlling activation of its progenitor, and perhaps other ANGPTLs. Inhibition of ANGPTL8 provides a new therapeutic strategy for reducing plasma lipoprotein levels.

Angiopoietin-like protein (ANGPTL)8 (alternatively called TD26, RIFL, Lipasin, and Betatrophin) is a newly recognized ANGPTL family member that has been implicated in both triglyceride (TG) and glucose metabolism. Hepatic overexpression of ANGPTL8 causes hypertriglyceridemia and increased insulin secretion. Here we examined the effects of inactivating Angptl8 on TG and glucose metabolism in mice. Angptl8 knockout (Angptl8 ⁻/⁻) mice gained weight more slowly than wild-type littermates due to a selective reduction in adipose tissue accretion. Plasma levels of TGs of the Angptl8 ⁻/⁻ mice were similar to wild-type animals in the fasted state but paradoxically decreased after refeeding. The lower TG levels were associated with both a reduction in very low density lipoprotein secretion and an increase in lipoprotein lipase (LPL) activity. Despite the increase in LPL activity, the uptake of very low density lipoprotein-TG is markedly reduced in adipose tissue but preserved in hearts of fed Angptl8 ⁻/⁻ mice. Taken together, these data indicate that ANGPTL8 plays a key role in the metabolic transition between fasting and refeeding; it is required to direct fatty acids to adipose tissue for storage in the fed state. Finally, glucose and insulin tolerance testing revealed no alterations in glucose homeostasis in mice fed either a chow or high fat diet. Thus, although absence of ANGPTL8 profoundly disrupts TG metabolism, we found no evidence that it is required for maintenance of glucose homeostasis.

The relative activity of lipoprotein lipase (LPL) in different tissues controls the partitioning of lipoprotein-derived 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.

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 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.

Two parallel pathways produce cholesterol: the Bloch and Kandutsch-Russell pathways. Here we used stable isotope labeling and isotopomer analysis to trace sterol flux through the two pathways in mice. Surprisingly, no tissue used the canonical K–R pathway. Rather, a hybrid pathway was identified that we call the modified K–R (MK–R) pathway. Proportional flux through the Bloch pathway varied from 8% in preputial gland to 97% in testes, and the tissue-specificity observed in vivo was retained in cultured cells. The distribution of sterol isotopomers in plasma mirrored that of liver. Sterol depletion in cultured cells increased flux through the Bloch pathway, whereas overexpression of 24-dehydrocholesterol reductase (DHCR24) enhanced usage of the MK–R pathway. Thus, relative use of the Bloch and MK–R pathways is highly variable, tissue-specific, flux dependent, and epigenetically fixed. Maintenance of two interdigitated pathways permits production of diverse bioactive sterols that can be regulated independently of cholesterol. Cholesterol is important for animals, both as an essential component of the membrane that surrounds cells and as a building block to make hormones and other biologically important molecules. However, cells limit how much cholesterol they make because an excess of this fatty molecule can cause serious health problems, including heart disease and stroke. Cholesterol is made via a complex process that involves more than 30 different steps, which can be organized into two biochemical pathways (named the Bloch pathway and the Kandutsch–Russell pathway). The enzymes that carry out the steps in these pathways have been characterized in detail. Less is known about which of the two pathways is actually used in different cells and tissues, or how much cholesterol each pathway produces. This is partly because it is difficult to distinguish between the closely related intermediate molecules that are formed in each pathway. Mitsche et al. have now used mass spectrometry and isotope labeling techniques to analyze the relative contributions of the two cholesterol-making pathways in both cells grown in the laboratory and in mice. The experiments show that many cells use the Bloch pathway. However, no cells were found to use the Kandutsch–Russell pathway as it was originally described. Rather, some of the cells used a hybrid pathway where the production of cholesterol was started using the Bloch pathway and then after a certain number of steps, the process switched to using part of the Kandutsch–Russell pathway. Mitsche et al. referred to this mixed system as the ‘modified Kandutsch–Russell pathway’. Mitsche et al. next examined the flow of molecules through these two pathways in different tissues and observed that the Bloch pathway is exclusively used in the testes and adrenal glands, which produce high levels of cholesterol. In contrast, the skin and brain use the modified Kandutsch–Russell pathway. In some tissues, a fraction of the building blocks that can be used to make cholesterol were instead diverted to make other products. This suggests that animals have maintained the two pathways over the course of evolution to enable them to generate a variety of products, which can be used to carry out different biological processes. One challenge following this work will be to use the newly developed methods to analyze other complex biochemical pathways.

Angiopoietin-like protein 3 (ANGPTL3) is a circulating inhibitor of lipoprotein and endothelial lipase whose physiological function has remained obscure. Here we show that ANGPTL3 plays a major role in promoting uptake of circulating very low density lipoprotein-triglycerides (VLDL-TGs) into white adipose tissue (WAT) rather than oxidative tissues (skeletal muscle, heart brown adipose tissue) in the fed state. This conclusion emerged from studies ofAngptl3−/− mice. Whereas feeding increased VLDL-TG uptake into WAT eightfold in wild-type mice, no increase occurred in fedAngptl3−/− animals. Despite the reduction in delivery to and retention of TG in WAT, fat mass was largely preserved by a compensatory increase in de novo lipogenesis inAngptl3−/− mice. Glucose uptake into WAT was increased 10-fold in KO mice, and tracer studies revealed increased conversion of glucose to fatty acids in WAT but not liver. It is likely that the increased uptake of glucose into WAT explains the increased insulin sensitivity associated with inactivation of ANGPTL3. The beneficial effects of ANGPTL3 deficiency on both glucose and lipoprotein metabolism make it an attractive therapeutic target.