Martha D. Wilson

Targeted Deletion of the Ileal Bile Acid Transporter Eliminates Enterohepatic Cycling of Bile Acids in Mice

The ileal apical sodium bile acid cotransporter participates in the enterohepatic circulation of bile acids. In patients with primary bile acid malabsorption, mutations in the ileal bile acid transporter gene (Slc10a2) lead to congenital diarrhea, steatorrhea, and reduced plasma cholesterol levels. To elucidate the quantitative role of Slc10a2 in intestinal bile acid absorption, the Slc10a2 gene was disrupted by homologous recombination in mice. Animals heterozygous (Slc10a2+/–) and homozygous (Slc10a2–/–) for this mutation were physically indistinguishable from wild type mice. In the Slc10a2–/– mice, fecal bile acid excretion was elevated 10- to 20-fold and was not further increased by feeding a bile acid binding resin. Despite increased bile acid synthesis, the bile acid pool size was decreased by 80% and selectively enriched in cholic acid in the Slc10a2–/– mice. On a low fat diet, the Slc10a2–/– mice did not have steatorrhea. Fecal neutral sterol excretion was increased only 3-fold, and intestinal cholesterol absorption was reduced only 20%, indicating that the smaller cholic acid-enriched bile acid pool was sufficient to facilitate intestinal lipid absorption. Liver cholesteryl ester content was reduced by 50% in Slc10a2–/– mice, and unexpectedly plasma high density lipoprotein cholesterol levels were slightly elevated. These data indicate that Slc10a2 is essential for efficient intestinal absorption of bile acids and that alternative absorptive mechanisms are unable to compensate for loss of Slc10a2 function.

Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol

We previously showed that macrophages from macrophage-specific ATP-binding cassette transporter A1 (ABCA1) knockout (Abca1-M/-M) mice had an enhanced proinflammatory response to the Toll-like receptor (TLR) 4 agonist, lipopolysaccharide (LPS), compared with wild-type (WT) mice. In the present study, we demonstrate a direct association between free cholesterol (FC), lipid raft content, and hyper-responsiveness of macrophages to LPS in WT mice. Abca1-M/-M macrophages were also hyper-responsive to specific agonists to TLR2, TLR7, and TLR9, but not TLR3, compared with WT macrophages. We hypothesized that ABCA1 regulates macrophage responsiveness to TLR agonists by modulation of lipid raft cholesterol and TLR mobilization to lipid rafts. We demonstrated that Abca1-M/-M vs. WT macrophages contained 23% more FC in isolated lipid rafts. Further, mass spectrometric analysis suggested raft phospholipid composition was unchanged. Although cell surface expression of TLR4 was similar between Abca1-M/-M and WT macrophages, significantly more TLR4 was distributed in membrane lipid rafts in Abca1-M/-M macrophages. Abca1-M/-M macrophages also exhibited increased trafficking of the predominantly intracellular TLR9 into lipid rafts in response to TLR9-specific agonist (CpG). Collectively, our data suggest that macrophage ABCA1 dampens inflammation by reducing MyD88-dependent TLRs trafficking to lipid rafts by selective reduction of FC content in lipid rafts.

Dietary Monounsaturated Fatty Acids Promote Aortic Atherosclerosis in LDL Receptor–Null, Human ApoB100–Overexpressing Transgenic Mice

In mice with genetically engineered high levels of plasma low density lipoprotein (LDL), we tested the hypothesis that an increase in the dietary content of monounsaturated fatty acids but not of polyunsaturated fatty acids would promote atherosclerosis. The mouse model used was an LDL receptor-null, human apoB100-overexpressing strain. Six experimental groups of 19 to 38 mice of both sexes were established when the animals had reached 8 weeks of age. For the next 16 weeks, individual groups were fed either a commercial diet or prepared diets including fat as 10% of energy, with 5 different fatty acid enrichment patterns including the following: saturated (sat), cis and trans monounsaturated (mono), and n-3 and n-6 polyunsaturated (poly). Highly significant differences (ANOVA, P<0. 0001) in LDL cholesterol (in mg/dL) were found, with the rank order at 16 weeks being trans mono (mean, 1390)>sat (922)=cis mono (869)=n-6 poly (868)>n-3 poly (652)>commercial diet (526). Significant elevations in very low density lipoprotein cholesterol were also found in the trans and cis mono and sat groups, and triacylglycerol concentrations were also elevated in all groups. High density lipoprotein cholesterol concentrations were consistently low (20 to 50 mg/dL) in all groups. Highly significant differences (ANOVA, P<0.0001) in atherosclerosis, quantified by measurement of aortic cholesteryl ester concentration (mg/g protein) among dietary fatty acid groups were found, with the order being trans mono (mean, 50.4)>sat (35.6)=cis mono (34.6)>n-6 poly (18. 3)=n-3 poly (9.7)=commercial diet (7.8). Therefore, in this mouse model of hypercholesterolemia, dietary cis or trans monounsaturated fat did not protect against atherosclerosis development, whereas aortic atherosclerosis in either of the polyunsaturated fat groups was significantly less than in the saturated fat group.

Deficiency of acyl CoA:cholesterol acyltransferase 2 prevents atherosclerosis in apolipoprotein E-deficient mice

Deficiency of acyl CoA:cholesterol acyltransferase 2 (ACAT2) in mice results in a reduction in cholesterol ester synthesis in the small intestine and liver, which in turn limits intestinal cholesterol absorption, hepatic cholesterol gallstone formation, and the accumulation of cholesterol esters in the plasma lipoproteins. Here we examined the contribution of ACAT2-derived cholesterol esters to atherosclerosis by crossing ACAT2-deficient (ACAT2–/–) mice with apolipoprotein (apo) E-deficient (ApoE–/–) mice, an atherosclerosis-susceptible strain that has impaired apoE-mediated clearance of apoB-containing lipoproteins. ACAT2–/– ApoE–/– mice and ACAT2+/+ ApoE–/– (control) mice had similar elevations of plasma apoB and total plasma lipids; however, the lipid cores of the apoB-containing lipoproteins in ACAT2–/– ApoE–/– mice contained primarily triglycerides rather than cholesterol esters. At 30 wk of age, only the control mice had significant atherosclerosis, which was nearly absent in ACAT2–/– ApoE–/– mice. ACAT2 deficiency in the apoE-deficient background also led to a compensatory increase in the activity of lecithin/cholesterol acyltransferase, the major plasma cholesterol esterification enzyme, which increased high-density lipoprotein cholesterol esters. Our results demonstrate the crucial role of ACAT2-derived cholesterol esters in the development of atherosclerosis in mice and suggest that triglyceride-rich apoB-containing lipoproteins are not as atherogenic as those containing cholesterol esters. Our results also support the rationale of pharmacological inhibition of ACAT2 as a therapy for atherosclerosis.

Inhibition of Stearoyl-Coenzyme A Desaturase 1 Dissociates Insulin Resistance and Obesity From Atherosclerosis

Background— Stearoyl-coenzyme A desaturase 1 (SCD1) is a well-known enhancer of the metabolic syndrome. The purpose of the present study was to investigate the role of SCD1 in lipoprotein metabolism and atherosclerosis progression. Methods and Results— Antisense oligonucleotides were used to inhibit SCD1 in a mouse model of hyperlipidemia and atherosclerosis (LDLr−/−Apob100/100). In agreement with previous reports, inhibition of SCD1 protected against diet-induced obesity, insulin resistance, and hepatic steatosis. Unexpectedly, however, SCD1 inhibition strongly promoted aortic atherosclerosis, which could not be reversed by dietary oleate. Further analyses revealed that SCD1 inhibition promoted accumulation of saturated fatty acids in plasma and tissues and reduced plasma triglyceride, yet had little impact on low-density lipoprotein cholesterol. Because dietary saturated fatty acids have been shown to promote inflammation through toll-like receptor 4, we examined macrophage toll-like receptor 4 function. Interestingly, SCD1 inhibition resulted in alterations in macrophage membrane lipid composition and marked hypersensitivity to toll-like receptor 4 agonists. Conclusions— This study demonstrates that atherosclerosis can occur independently of obesity and insulin resistance and argues against SCD1 inhibition as a safe therapeutic target for the metabolic syndrome.

CGI-58 knockdown in mice causes hepatic steatosis, but prevents diet-induced obesity and glucose intolerance

Mutations of Comparative Gene Identification-58 (CGI-58) in humans cause triglyceride (TG) accumulation in multiple tissues. Mice genetically lacking CGI-58 die shortly after birth due to a skin barrier defect. To study the role of CGI-58 in integrated lipid and energy metabolism, we utilized antisense oligonucleotides (ASOs) to inhibit CGI-58 expression in adult mice. Treatment with two distinct CGI-58-targeting ASOs resulted in ∼80–95% knockdown of CGI-58 protein expression in both liver and white adipose tissue. In chow-fed mice, ASO-mediated depletion of CGI-58 did not alter weight gain, plasma TG, or plasma glucose, yet raised hepatic TG levels ∼4-fold. When challenged with a high-fat diet (HFD), CGI-58 ASO-treated mice were protected against diet-induced obesity, but their hepatic contents of TG, diacylglycerols, and ceramides were all elevated, and intriguingly, their hepatic phosphatidylglycerol content was increased by 10-fold. These hepatic lipid alterations were associated with significant decreases in hepatic TG hydrolase activity, hepatic lipoprotein-TG secretion, and plasma concentrations of ketones, nonesterified fatty acids, and insulin. Additionally, HFD-fed CGI-58 ASO-treated mice were more glucose tolerant and insulin sensitive. Collectively, this work demonstrates that CGI-58 plays a critical role in limiting hepatic steatosis and maintaining hepatic glycerophospholipid homeostasis and has unmasked an unexpected role for CGI-58 in promoting HFD-induced obesity and insulin resistance.

Defining the atherogenicity of large and small lipoproteins containing apolipoprotein B100

Apo-E-deficient apo-B100-only mice (APOE:(-/-)APOB:(100/100)) and LDL receptor-deficient apo-B100-only mice (LDLR:(-/-)APOB:(100/100)) have similar total plasma cholesterol levels, but nearly all of the plasma cholesterol in the former animals is packaged in VLDL particles, whereas, in the latter, plasma cholesterol is found in smaller LDL particles. We compared the apo-B100-containing lipoprotein populations in these mice to determine their relation to susceptibility to atherosclerosis. The median size of the apo-B100-containing lipoprotein particles in APOE:(-/-)APOB:(100/100) plasma was 53.4 nm versus only 22.1 nm in LDLR:(-/-)APOB:(100/100) plasma. The plasma levels of apo-B100 were three- to fourfold higher in LDLR:(-/-)APOB:(100/100) mice than in APOE:(-/-)APOB:(100/100) mice. After 40 weeks on a chow diet, the LDLR:(-/-)APOB:(100/100) mice had more extensive atherosclerotic lesions than APOE:(-/-)APOB:(100/100) mice. The aortic DNA synthesis rate and the aortic free and esterified cholesterol contents were also higher in the LDLR:(-/-)APOB:(100/100) mice. These findings challenge the notion that all non-HDL lipoproteins are equally atherogenic and suggest that at a given cholesterol level, large numbers of small apo-B100-containing lipoproteins are more atherogenic than lower numbers of large apo-B100-containing lipoproteins.

Dietary cholesterol and downregulation of cholesterol 7 alpha-hydroxylase and cholesterol absorption in African green monkeys.

In this study, hepatic production of bile acid was considered together with intestinal cholesterol absorption as potential regulatory sites responsive to dietary cholesterol. Sequential liver biopsies were taken from 45 feral African green monkeys studied during three different diet periods. Low-fat Monkey Chow was fed during the baseline period, a cholesterol and fat-enriched diet was then fed for 12 wk during period 2, and finally, after a washout period of 10 wk, three subgroups were fed low-, moderate-, and high-cholesterol diets for 12 mo during period 3. The percentage of cholesterol absorbed in the intestine was significantly lower when higher levels of cholesterol were fed; however, this percentage was significantly and positively correlated to plasma cholesterol concentration at each dietary cholesterol level. Hepatic free and esterified cholesterol content were significantly elevated by dietary cholesterol challenge and remained elevated even after 20 wk of low-cholesterol diets. Hepatic mRNA abundance for cholesterol 7 alpha-hydroxylase (C7H) was significantly lower (approximately 60%) when the high-cholesterol diet was fed, with the decrease being greater than that seen for low density lipoprotein (LDL) receptor mRNA. At the same time, hepatic mRNA abundance for apolipoprotein B and hepatic lipase were not diet sensitive. C7H activity was decreased to a similar extent by diet as was C7H mRNA, although the correlation between enzyme activity and mRNA abundance was only r = 0.5, suggesting that dietary regulation includes factors in addition to transcriptional regulation. Activity and mRNA abundance of C7H remained decreased when liver esterified cholesterol content was reduced to only a two- to three-fold elevation over baseline, at a time when plasma cholesterol and hepatic LDL receptor mRNA abundance had returned to baseline levels. These data on liver C7H, obtained in one of the few primate species predisposed to cholesterol gallstone formation, support the hypothesis that the liver may attempt to downregulate intestinal cholesterol absorption by decreasing bile acid production when increased amounts of absorbed dietary cholesterol reach the liver. Presumably this represents attempted downregulation of intestinal cholesterol absorption by limiting bile acid availability as a means to maintain hepatic cholesterol balance.

Targeted Depletion of Hepatic ACAT2-driven Cholesterol Esterification Reveals a Non-biliary Route for Fecal Neutral Sterol Loss

Deletion of acyl-CoA:cholesterol O-acyltransferase 2 (ACAT2) in mice results in resistance to diet-induced hypercholesterolemia and protection against atherosclerosis. Recently, our group has shown that liver-specific inhibition of ACAT2 via antisense oligonucleotide (ASO)-mediated targeting likewise limits atherosclerosis. However, whether this atheroprotective effect was mediated by: 1) prevention of packaging of cholesterol into apoB-containing lipoproteins, 2) augmentation of nascent HDL cholesterol secretion, or 3) increased hepatobiliary sterol secretion was not examined. Therefore, the purpose of these studies was to determine whether hepatic ACAT2 is rate-limiting in all three of these important routes of cholesterol homeostasis. Liver-specific depletion of ACAT2 resulted in reduced packaging of cholesterol into apoB-containing lipoproteins (very low density lipoprotein, intermediate density lipoprotein, and low density lipoprotein), whereas high density lipoprotein cholesterol levels remained unchanged. In the liver of ACAT2 ASO-treated mice, cholesterol ester accumulation was dramatically reduced, yet there was no reciprocal accumulation of unesterified cholesterol. Paradoxically, ASO-mediated depletion of hepatic ACAT2 promoted fecal neutral sterol excretion without altering biliary sterol secretion. Interestingly, during isolated liver perfusion, ACAT2 ASO-treated livers had augmented secretion rates of unesterified cholesterol and phospholipid. Furthermore, we demonstrate that liver-derived cholesterol from ACAT2 ASO-treated mice is preferentially delivered to the proximal small intestine as a precursor to fecal excretion. Collectively, these studies provide the first insight into the hepatic itinerary of cholesterol when cholesterol esterification is inhibited only in the liver, and provide evidence for a novel non-biliary route of fecal sterol loss.

Characterization and Heritability of Obesity and Associated Risk Factors in Vervet Monkeys

Objective: The objective was to determine the prevalence and heritability of obesity and risk factors associated with metabolic syndrome (MS) in a pedigreed colony of vervet monkeys.