Nicholas O. Davidson

Specific Expression of Activation-induced Cytidine Deaminase (AID), a Novel Member of the RNA-editing Deaminase Family in Germinal Center B Cells

We have identified a novel gene referred to asactivation-induced deaminase (AID) by subtraction of cDNAs derived from switch-induced and uninduced murine B lymphoma CH12F3-2 cells, more than 80% of which switch exclusively to IgA upon stimulation. The amino acid sequence encoded by AID cDNA is homologous to that of apolipoprotein B (apoB) mRNA-editing enzyme, catalytic polypeptide 1 (APOBEC-1), a type of cytidine deaminase that constitutes a catalytic subunit for the apoB mRNA-editing complex. In vitro experiments using a glutathione S-transferase AID fusion protein revealed significant cytidine deaminase activity that is blocked by tetrahydrouridine and by zinc chelation. However, AID alone did neither demonstrate activity in C to U editing of apoB mRNA nor bind to AU-rich RNA targets. AID mRNA expression is induced in splenic B cells that were activated in vitro or by immunizations with sheep red blood cells. In situ hybridization of immunized spleen sections revealed the restricted expression of AID mRNA in developing germinal centers in which modulation of immunoglobulin gene information through somatic hypermutation and class switch recombination takes place. Taken together, these findings suggest that AID is a new member of the RNA-editing deaminase family and may play a role in genetic events in the germinal center B cell.

Cytidine Deamination of Retroviral DNA by Diverse APOBEC Proteins

The human cytidine deaminase APOBEC3G edits both nascent human immunodeficiency virus (HIV) and murine leukemia virus (MLV) reverse transcripts, resulting in loss of infectivity. The HIV Vif protein is able to protect both viruses from this innate restriction to infection. Here, we demonstrate that a number of other APOBEC family members from both humans and rodents can mediate anti-HIV effects, through cytidine deamination. Three of these, rat APOBEC1, mouse APOBEC3, and human APOBEC3B, are able to inhibit HIV infectivity even in the presence of Vif. Like APOBEC3G, human APOBEC3F preferentially restricts vif-deficient virus. Indeed, the mutation spectra and expression profile found for APOBEC3F indicate that this enzyme, together with APOBEC3G, accounts for the G to A hypermutation of proviruses described in HIV-infected individuals. Surprisingly, although MLV infectivity is acutely reduced by APOBEC3G, no other family member tested here had this effect. It is especially interesting that although both rodent APOBECs markedly diminish wild-type HIV infectivity, MLV is resistant to these proteins. This implies that MLV may have evolved to avoid deamination by mouse APOBEC3. Overall, our findings show that although APOBEC family members are highly related, they exhibit significantly distinct antiviral characteristics that may provide new insights into host-pathogen interactions.

Troglitazone action is independent of adipose tissue.

We have investigated the antidiabetic action of troglitazone in aP2/DTA mice, whose white and brown fat was virtually eliminated by fat-specific expression of diphtheria toxin A chain. aP2/DTA mice had markedly suppressed serum leptin levels and were hyperphagic, but did not gain excess weight. aP2/DTA mice fed a control diet were hyperlipidemic, hyperglycemic, and had hyperinsulinemia indicative of insulin-resistant diabetes. Treatment with troglitazone alleviated the hyperglycemia, normalized the tolerance to intraperitoneally injected glucose, and significantly decreased elevated insulin levels. Troglitazone also markedly decreased the serum levels of cholesterol, triglycerides, and free fatty acids both in wild-type and aP2/DTA mice. The decrease in serum triglycerides in aP2/DTA mice was due to a marked reduction in VLDL- and LDL-associated triglyceride. In skeletal muscle, triglyceride levels were decreased in aP2/DTA mice compared with controls, but glycogen levels were increased. Troglitazone treatment decreased skeletal muscle, but not hepatic triglyceride and increased hepatic and muscle glycogen content in wild-type mice. Troglitazone decreased muscle glycogen content in aP2/DTA mice without affecting muscle triglyceride levels. The levels of peroxisomal proliferator-activated receptor gamma mRNA in liver increased slightly in aP2/DTA mice and were not changed by troglitazone treatment. The results demonstrate that insulin resistance and diabetes can occur in animals without significant adipose deposits. Furthermore, troglitazone can alter glucose and lipid metabolism independent of its effects on adipose tissue.

Decreased Hepatic Triglyceride Accumulation and Altered Fatty Acid Uptake in Mice with Deletion of the Liver Fatty Acid-binding Protein Gene

Liver fatty acid-binding protein (L-Fabp) is an abundant cytosolic lipid-binding protein with broad substrate specificity, expressed in mammalian enterocytes and hepatocytes. We have generated mice with a targeted deletion of the endogenous L-Fabp gene and have characterized their response to alterations in hepatic fatty acid flux following prolonged fasting. Chow-fed L-Fabp–/– mice were indistinguishable from wild-type littermates with regard to growth, serum and tissue lipid profiles, and fatty acid distribution within hepatic complex lipid species. In response to 48-h fasting, however, wild-type mice demonstrated a ∼10-fold increase in hepatic triglyceride content while L-Fabp–/– mice demonstrated only a 2-fold increase. Hepatic VLDL secretion was decreased in L-Fabp–/– mice suggesting that the decreased accumulation of hepatic triglyceride was not the result of increased secretion. Fatty acid oxidation, as inferred from serum β-hydroxybutyrate levels, was increased in response to fasting, although the increase in L-Fabp–/– mice was significantly reduced in comparison to wild-type controls, despite comparable induction of PPARα target genes. Studies in primary hepatocytes revealed indistinguishable initial rates of oleate uptake, but longer intervals revealed reduced rates of uptake in fasted L-Fabp–/– mice. Oleate incorporation into cellular triglyceride and diacylglycerol was reduced in L-Fabp–/– mice although incorporation into phospholipid and cholesterol ester was no different than wild-type controls. These data point to an inducible defect in fatty acid utilization in fasted L-Fabp–/– mice that involves targeting of substrate for use in triglyceride metabolism.

Role of the Gut in Lipid Homeostasis

Intestinal lipid transport plays a central role in fat homeostasis. Here we review the pathways regulating intestinal absorption and delivery of dietary and biliary lipid substrates, principally long-chain fatty acid, cholesterol, and other sterols. We discuss the regulation and functions of CD36 in fatty acid absorption, NPC1L1 in cholesterol absorption, as well as other lipid transporters including FATP4 and SRB1. We discuss the pathways of intestinal sterol efflux via ABCG5/G8 and ABCA1 as well as the role of the small intestine in high-density lipoprotein (HDL) biogenesis and reverse cholesterol transport. We review the pathways and genetic regulation of chylomicron assembly, the role of dominant restriction points such as microsomal triglyceride transfer protein and apolipoprotein B, and the role of CD36, l-FABP, and other proteins in formation of the prechylomicron complex. We will summarize current concepts of regulated lipoprotein secretion (including HDL and chylomicron pathways) and include lessons learned from families with genetic mutations in dominant pathways (i.e., abetalipoproteinemia, chylomicron retention disease, and familial hypobetalipoproteinemia). Finally, we will provide an integrative view of intestinal lipid homeostasis through recent findings on the role of lipid flux and fatty acid signaling via diverse receptor pathways in regulating absorption and production of satiety factors.

CD36 deficiency impairs intestinal lipid secretion and clearance of chylomicrons from the blood

CD36 mediates the transfer of fatty acids (FAs) across the plasma membranes of muscle and adipose cells, thus playing an important role in regulating peripheral FA metabolism in vivo. In the proximal intestine, CD36 is localized in abundant quantities on the apical surface of epithelial cells, a pattern similar to that of other proteins implicated in the uptake of dietary FAs. To define the role of CD36 in the intestine, we examined FA utilization and lipoprotein secretion by WT and CD36-null mice in response to acute and chronic fat feeding. CD36-null mice given a fat bolus by gavage or fed a high-fat diet accumulated neutral lipid in the proximal intestine, which indicated abnormal lipid processing. Using a model in which mice were equipped with lymph fistulae, we obtained evidence of defective lipoprotein secretion by directly measuring lipid output. The secretion defect appeared to reflect an impaired ability of CD36-null enterocytes to efficiently synthesize triacylglycerols from dietary FAs in the endoplasmic reticulum. In the plasma of intact mice, the reduced intestinal lipid secretion was masked by slow clearance of intestine-derived lipoproteins. The impaired clearance occurred despite normal lipoprotein lipase activity and likely reflected feedback inhibition of the lipase by FAs due to their defective removal from the plasma. We conclude that CD36 is important for both secretion and clearance of intestinal lipoproteins. CD36 deficiency results in hypertriglyceridemia both in the postprandial and fasting states and in humans may constitute a risk factor for diet-induced type 2 diabetes and cardiovascular disease.

Protection against Western diet–induced obesity and hepatic steatosis in liver fatty acid–binding protein knockout mice†

Liver fatty acid–binding protein (L‐Fabp) regulates murine hepatic fatty acid trafficking in response to fasting. In this study, we show that L‐Fabp−/− mice fed a high‐fat Western diet for up to 18 weeks are less obese and accumulate less hepatic triglyceride than C57BL/6J controls. Paradoxically, both control and L‐Fabp−/− mice manifested comparable glucose intolerance and insulin resistance when fed a Western diet. Protection against obesity in Western diet–fed L‐Fabp−/− mice was not due to discernable changes in food intake, fat malabsorption, or heat production, although intestinal lipid secretion kinetics were significantly slower in both chow‐fed and Western diet–fed L‐Fabp−/− mice. By contrast, there was a significant increase in the respiratory exchange ratio in L‐Fabp−/− mice, suggesting a shift in energy substrate use from fat to carbohydrate, findings supported by an approximately threefold increase in serum lactate. Microarray analysis revealed increased expression of genes involved in lipid synthesis (fatty acid synthase, squalene epoxidase, hydroxy‐methylglutaryl coenzyme A reductase), while genes involved in glycolysis (glucokinase and glycerol kinase) were decreased in L‐Fabp−/− mice. Fatty acid synthase expression was also increased in the skeletal muscle of L‐Fabp−/− mice. In conclusion, L‐Fabp may function as a metabolic sensor in regulating lipid homeostasis. We suggest that L‐Fabp−/− mice are protected against Western diet–induced obesity and hepatic steatosis through a series of adaptations in both hepatic and extrahepatic energy substrate use. (HEPATOLOGY 2006;44:1191–1205.)

C-to-U RNA Editing: Mechanisms Leading to Genetic Diversity

Substitutional RNA Editing: Biochemical Mechanisms and Targets for C-to-U RNA Editing in Mammals RNA editing is an important mechanism for regulating genetic plasticity through the generation of alternative protein products from a single structural gene. Substitutional RNA editing employs a variety of genetic mechanisms, the biochemical basis of which has been elucidated following the development of in vitro assays that recapitulate important elements of this process. Two types of substitutional RNA exist in mammals, namely A-to-I and C-to-U RNA editing (1, 2). Important biochemical distinctions between these two processes provide an informative basis for understanding the mechanisms of C-to-U RNA editing and the adaptations that control target specificity. A-to-I RNA editing is mediated by a family of adenosine deaminases acting on double-stranded RNA (ADARs) with partially overlapping target specificity (1, 2). The absolute requirement for a double-stranded RNA template distinguishes A-to-I and C-to-U RNA editing because the former requires a pre-mRNA template containing intronic regions and is thus biochemically confined to unspliced transcripts. A further distinction biochemically is that ADAR enzymes do not require additional cofactors. ADARs contain both double-stranded RNA binding domains and a deaminase domain and function as modular editing enzymes (2, 3). The best characterized example of C-to-U RNA editing involves the nuclear transcript encoding intestinal apolipoprotein B (apoB) (4). ApoB RNA editing changes a CAA to a UAA stop codon, generating a truncated protein, apoB48 (4). ApoB RNA editing has important effects on lipoprotein metabolism, and its emergence defines distinct pathways for intestinal and hepatic lipid transport in mammals (4). C-to-U editing of apoB RNA requires a single-strand template (Fig. 1) with well defined characteristics in the immediate vicinity of the edited base, as well as protein cofactors that assemble into a functional complex referred to as a holoenzyme or editosome. This functional complex includes a minimal core composed of apobec-1, the catalytic deaminase, and a competence factor, apobec-1 complementation factor (ACF), that functions as an adaptor protein by binding both the deaminase and the RNA substrate (Fig. 1). The interaction of these protein components and their higher order interactions with the nuclear transcript illustrates the complexity of site-selectivity in C-to-U RNA editing. A second example of C-to-U RNA editing in mammals involves site-specific deamination of a CGA to UGA codon in the neurofibromatosis type 1 (NF1) mRNA (5). NF1 RNA editing generates a translational termination codon at position 3916 that is predicted to truncate the protein product neurofibromin at the 5 end of a critical domain (6) involved in GTPase activation (Fig. 2). Unlike apoB RNA editing, there is no formal proof that a truncated protein is generated. This example of C-to-U RNA editing has been demonstrated in peripheral nerve sheath tumors from patients with NF1 and may share elements of the same machinery as apoB RNA editing, as discussed below. A third target for C-to-U editing, NAT1, was revealed following forced transgenic overexpression of apobec-1 in murine and rabbit hepatocytes (7). NAT1 is homologous to the translational repressor eIF4G and undergoes C-to-U editing at multiple sites, with the creation of stop codons that in turn reduce protein abundance (7).

Mutations in the K-ras oncogene induced by 1,2-dimethylhydrazine in preneoplastic and neoplastic rat colonic mucosa.

These experiments were conducted to determine whether point mutations activating K-ras or H-ras oncogenes, induced by the procarcinogen 1,2-dimethylhydrazine (DMH), were detectable in preneoplastic or neoplastic rat colonic mucosa. Rats were injected weekly with diluent or DMH at 20 mg/kg body wt for 5, 10, 15, or 25 wk, killed, and their colons dissected. DNA was extracted from diluent-injected control animals, histologically normal colonic mucosa from carcinogen-treated animals, and from carcinomas. Ras mutations were characterized by differential hybridization using allele-specific oligonucleotide probes to polymerase chain reaction--amplified DNA, and confirmed by DNA sequencing. While no H-ras mutations were detectable in any group, K-ras (G to A) mutations were found in 66% of DMH-induced colon carcinomas. These mutations were at the second nucleotide of codons 12 or 13 or the first nucleotide of codon 59 of the K-ras gene. The same type of K-ras mutations were observed in premalignant colonic mucosa from 2 out of 11 rats as early as 15 wk after beginning carcinogen injections when no dysplasia, adenomas, or carcinomas were histologically evident, suggesting that ras mutation may be an early event in colon carcinogenesis.

Protective mucosal immunity mediated by epithelial CD1d and IL-10

The mechanisms by which mucosal homeostasis is maintained are of central importance to inflammatory bowel disease. Critical to these processes is the intestinal epithelial cell (IEC), which regulates immune responses at the interface between the commensal microbiota and the host. CD1d presents self and microbial lipid antigens to natural killer T (NKT) cells, which are involved in the pathogenesis of colitis in animal models and human inflammatory bowel disease. As CD1d crosslinking on model IECs results in the production of the important regulatory cytokine interleukin (IL)-10 (ref. 9), decreased epithelial CD1d expression—as observed in inflammatory bowel disease—may contribute substantially to intestinal inflammation. Here we show in mice that whereas bone-marrow-derived CD1d signals contribute to NKT-cell-mediated intestinal inflammation, engagement of epithelial CD1d elicits protective effects through the activation of STAT3 and STAT3-dependent transcription of IL-10, heat shock protein 110 (HSP110; also known as HSP105), and CD1d itself. All of these epithelial elements are critically involved in controlling CD1d-mediated intestinal inflammation. This is demonstrated by severe NKT-cell-mediated colitis upon IEC-specific deletion of IL-10, CD1d, and its critical regulator microsomal triglyceride transfer protein (MTP), as well as deletion of HSP110 in the radioresistant compartment. Our studies thus uncover a novel pathway of IEC-dependent regulation of mucosal homeostasis and highlight a critical role of IL-10 in the intestinal epithelium, with broad implications for diseases such as inflammatory bowel disease.