Jay D. Horton

SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver

Lipid homeostasis in vertebrate cells is regulated by a family of membrane-bound transcription factors designated sterol regulatory element–binding proteins (SREBPs). SREBPs directly activate the expression of more than 30 genes dedicated to the synthesis and uptake of cholesterol, fatty acids, triglycerides, and phospholipids, as well as the NADPH cofactor required to synthesize these molecules (1–4). In the liver, three SREBPs regulate the production of lipids for export into the plasma as lipoproteins and into the bile as micelles. The complex, interdigitated roles of these three SREBPs have been dissected through the study of ten different lines of gene-manipulated mice. These studies form the subject of this review.

Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity.

Despite the increasing prevalence of nonalcoholic fatty liver disease (NAFLD), its pathogenesis and clinical significance remain poorly defined. In this study, we examined and compared the distribution of hepatic triglyceride content (HTGC) in 2,287 subjects from a multiethnic, population‐based sample (32.1% white, 48.3% black, and 17.5% Hispanic) using proton magnetic resonance spectroscopy. HTGC varied over a wide range (0.0%‐41.7%; median, 3.6%) in the population. Almost one third of the population had hepatic steatosis, and most subjects with hepatic steatosis had normal levels of serum alanine aminotransferase (79%). The frequency of hepatic steatosis varied significantly with ethnicity (45% in Hispanics; 33% in whites; 24% in blacks) and sex (42% in white men; 24% in white women). The higher prevalence of hepatic steatosis in Hispanics was due to the higher prevalence of obesity and insulin resistance in this ethnic group. However, the lower frequency of hepatic steatosis in blacks was not explained by ethnic differences in body mass index, insulin resistance, ethanol ingestion, or medication use. The prevalence of hepatic steatosis was greater in men than women among whites, but not in blacks or Hispanics. The ethnic differences in the frequency of hepatic steatosis in this study mirror those observed previously for NAFLD‐related cirrhosis (Hispanics > whites > blacks). In conclusion, the significant ethnic and sex differences in the prevalence of hepatic steatosis documented in this study may have a profound impact on susceptibility to steatosis‐related liver disease. (HEPATOLOGY 2004;40:1387–1395.)

Molecular mediators of hepatic steatosis and liver injury

Obesity and its associated comorbidities are among the most prevalent and challenging conditions confronting the medical profession in the 21st century. A major metabolic consequence of obesity is insulin resistance, which is strongly associated with the deposition of triglycerides in the liver. Hepatic steatosis can either be a benign, noninflammatory condition that appears to have no adverse sequelae or can be associated with steatohepatitis: a condition that can result in end-stage liver disease, accounting for up to 14% of liver transplants in the US. Here we highlight recent advances in our understanding of the molecular events contributing to hepatic steatosis and nonalcoholic steatohepatitis.

Human Fatty Liver Disease: Old Questions and New Insights

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.

Combined analysis of oligonucleotide microarray data from transgenic and knockout mice identifies direct SREBP target genes

The synthesis of fatty acids and cholesterol, the building blocks of membranes, is regulated by three membrane-bound transcription factors: sterol regulatory element-binding proteins (SREBP)-1a, -1c, and -2. Their function in liver has been characterized in transgenic mice that overexpress each SREBP isoform and in mice that lack all three nuclear SREBPs as a result of gene knockout of SREBP cleavage-activating protein (SCAP), a protein required for nuclear localization of SREBPs. Here, we use oligonucleotide arrays hybridized with RNA from livers of three lines of mice (transgenic for SREBP-1a, transgenic for SREBP-2, and knockout for SCAP) to identify genes that are likely to be direct targets of SREBPs in liver. A total of 1,003 genes showed statistically significant increased expression in livers of transgenic SREBP-1a mice, 505 increased in livers of transgenic SREBP-2 mice, and 343 showed decreased expression in Scap–/– livers. A subset of 33 genes met the stringent combinatorial criteria of induction in both SREBP transgenics and decreased expression in SCAP-deficient mice. Of these 33 genes, 13 were previously identified as direct targets of SREBP action. Of the remaining 20 genes, 13 encode enzymes or carrier proteins involved in cholesterol metabolism, 3 participate in fatty acid metabolism, and 4 have no known connection to lipid metabolism. Through application of stringent combinatorial criteria, the transgenic/knockout approach allows identification of genes whose activities are likely to be controlled directly by one family of transcription factors, in this case the SREBPs.

Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a.

The NH2-terminal domain of sterol-regulatory element binding protein-1a (SREBP-1a) activates transcription of genes encoding enzymes of cholesterol and fatty acid biosynthesis in cultured cells. This domain is synthesized as part of a membrane-bound precursor that is attached to the nuclear envelope and endoplasmic reticulum. In sterol-depleted cells a two-step proteolytic process releases this NH2-terminal domain, which enters the nucleus and activates transcription. Proteolysis is suppressed by sterols, thereby suppressing transcription. In the current experiments we produce transgenic mice that overexpress a truncated version of human SREBP-1a that includes the NH2-terminal domain but lacks the membrane attachment site. This protein enters the nucleus without a requirement for proteolysis, and therefore it cannot be down-regulated. Expression was driven by the phosphoenolpyruvate carboxykinase (PEPCK) promoter, which gives high level expression in liver. When placed on a low carbohydrate/high protein diet to induce the PEPCK promoter, the transgenic mice developed progressive and massive enlargement of the liver, owing to the engorgement of hepatocytes with cholesterol and triglycerides. The mRNAs encoding 3-hydroxy-3-methylglutaryl CoA (HMG CoA) synthase, HMG CoA reductase, squalene synthase, acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase-1 were all elevated markedly, as was the LDL receptor mRNA. The rates of cholesterol and fatty acid synthesis in liver were elevated 5- and 25-fold, respectively. Remarkably, plasma lipid levels were not elevated. The amount of white adipose tissue decreased progressively as the liver enlarged. These studies indicate that the NH2-terminal domain of SREBP-1a can produce major effects on lipid synthesis and storage in the liver.

Isoform 1c of sterol regulatory element binding protein is less active than isoform 1a in livers of transgenic mice and in cultured cells.

We have produced transgenic mice whose livers express a dominant positive NH2-terminal fragment of sterol regulatory element binding protein-1c (SREBP-1c). Unlike full-length SREBP-1c, the NH2-terminal fragment enters the nucleus without a requirement for proteolytic release from cell membranes, and hence it is immune to downregulation by sterols. We compared SREBP-1c transgenic mice with a line of transgenic mice that produces an equal amount of the NH2-terminal fragment of SREBP-1a. SREBP-1a and -1c are alternate transcripts from a single gene that differ in the first exon, which encodes part of an acidic activation domain. The 1a protein contains a long activation domain with 12 negatively charged amino acids, whereas the 1c protein contains a short activation domain with only 6 such amino acids. As previously reported, livers of the SREBP-1a transgenic mice were massively enlarged, owing to accumulation of triglycerides and cholesterol. SREBP-1c transgenic livers were only slightly enlarged with only a moderate increase in triglycerides, but not cholesterol. The mRNAs for the LDL receptor and several cholesterol biosynthetic enzymes were elevated in SREBP-la transgenic mice, but not in 1c transgenic mice. The mRNAs for fatty acid synthase and acetyl CoA carboxylase were elevated 9- and 16-fold in la animals, but only 2- and 4-fold in 1c animals. Experiments with transfected cells confirmed that SREBP-1c is a much weaker activator of transcription than SREBP-1a when both are expressed at levels approximating those found in nontransfected cells. SREBP-1c became a strong activator only when expressed at supraphysiologic levels. We conclude that SREBP-1a is the most active form of SREBP-1 and that SREBP-1c may be produced when cells require a lower rate of transcription of genes regulating cholesterol and fatty acid metabolism.

Activation of cholesterol synthesis in preference to fatty acid synthesis in liver and adipose tissue of transgenic mice overproducing sterol regulatory element-binding protein-2.

We produced transgenic mice that express a dominant-positive truncated form of sterol regulatory element-binding protein-2 (SREBP-2) in liver and adipose tissue. The encoded protein lacks the membrane-binding and COOH-terminal regulatory domains, and it is therefore not susceptible to negative regulation by cholesterol. Livers from the transgenic mice showed increases in mRNAs encoding multiple enzymes of cholesterol biosynthesis, the LDL receptor, and fatty acid biosynthesis. The elevations in mRNA for 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and HMG CoA reductase were especially marked (13-fold and 75-fold, respectively). As a result, the transgenic livers showed a 28-fold increase in the rate of cholesterol synthesis and a lesser fourfold increase in fatty acid synthesis, as measured by intraperitoneal injection of [3H]water. These results contrast with previously reported effects of dominant-positive SREBP-1a, which activated fatty acid synthesis more than cholesterol synthesis. In adipose tissue of the SREBP-2 transgenics, the mRNAs for cholesterol biosynthetic enzymes were elevated, but the mRNAs for fatty acid biosynthetic enzymes were not. We conclude that SREBP-2 is a relatively selective activator of cholesterol synthesis, as opposed to fatty acid synthesis, in liver and adipose tissue of mice.

Overexpression of ABCG5 and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorption of dietary cholesterol

Two ATP-binding cassette (ABC) transporters, ABCG5 and ABCG8, have been proposed to limit sterol absorption and to promote biliary sterol excretion in humans. To test this hypothesis, a P1 clone containing the human ABCG5 and ABCG8 genes was used to generate transgenic mice. The transgenes were expressed primarily in the liver and small intestine, mirroring the expression pattern of the endogenous genes. Transgene expression only modestly affected plasma and liver cholesterol levels but profoundly altered cholesterol transport. The fractional absorption of dietary cholesterol was reduced by about 50%, and biliary cholesterol levels were increased more than fivefold. Fecal neutral sterol excretion was increased three- to sixfold and hepatic cholesterol synthesis increased two- to fourfold in the transgenic mice. No significant changes in the pool size, composition, and fecal excretion of bile acids were observed in the transgenic mice. Transgene expression attenuated the increase in hepatic cholesterol content induced by consumption of a high cholesterol diet. These results demonstrate that increased expression of ABCG5 and ABCG8 selectively drives biliary neutral sterol secretion and reduces intestinal cholesterol absorption, leading to a selective increase in neutral sterol excretion and a compensatory increase in cholesterol synthesis.

Binding of Proprotein Convertase Subtilisin/Kexin Type 9 to Epidermal Growth Factor-like Repeat A of Low Density Lipoprotein Receptor Decreases Receptor Recycling and Increases Degradation

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes degradation of hepatic low density lipoprotein receptors (LDLR), the major route of clearance of circulating cholesterol. Gain-of-function mutations in PCSK9 cause hypercholesterolemia and premature atherosclerosis, whereas loss-of-function mutations result in hypocholesterolemia and protection from heart disease. Recombinant human PCSK9 binds the LDLR on the surface of cultured hepatocytes and promotes degradation of the receptor after internalization. Here we localized the site of binding of PCSK9 within the extracellular domain of the LDLR and determined the fate of the receptor after PCSK9 binding. Recombinant human PCSK9 interacted in a sequence-specific manner with the first epidermal growth factor-like repeat (EGF-A) in the EGF homology domain of the human LDLR. Similar binding specificity was observed between PCSK9 and purified EGF-A. Binding to EGF-A was calcium-dependent and increased dramatically with reduction in pH from 7 to 5.2. The addition of PCSK9, but not heat-inactivated PCSK9, to the medium of cultured hepatocytes resulted in redistribution of the receptor from the plasma membrane to lysosomes. These data are consistent with a model in which PCSK9 binding to EGF-A interferes with an acid-dependent conformational change required for receptor recycling. As a consequence, the LDLR is rerouted from the endosome to the lysosome where it is degraded.