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To the Editor:

Nonalcoholic fatty liver disease (NAFLD) is a burgeoning problem in developed countries and affects up to one-third of the population.1 NAFLD is considered to be a component of the metabolic syndrome; obesity is the primary risk factor, and weight loss and treatment of associated conditions (i.e., diabetes, hyperlipidemia, among others) are the only recommended therapies.2 Several recent studies in animal models and in humans have suggested that ezetimibe, a cholesterol-lowering agent that acts by inhibiting cholesterol absorption, may be an effective therapy for NAFLD.3-5 The most striking and consistent finding of these small, primarily open-label studies is a significant reduction in hepatic triglyceride content. Why inhibition of intestinal cholesterol absorption should impact hepatic triglyceride metabolism is unclear. Ezetimibe acts by inhibiting Nieman Pick C1-Like 1 (NPC1L1).6 Genetic deletion of NPC1L1 in mice decreases hepatic de novo lipogenesis. Therefore, ezetimibe may attenuate hepatic steatosis by limiting the synthesis of fatty acids in liver.7

DNA sequencing revealed that nonsynonymous (NS) sequence variants in NPC1L1 that confer a reduced capacity for intestinal cholesterol absorption are collectively common in the population, particularly among blacks.8, 9 Individuals who were heterozygous for one of the sequence variations in NPC1L1 had evidence of reduced sterol absoption and a 9% reduction in plasma low-density lipoprotein cholesterol. Inasmuch as these subjects represent a life-long genetic knockdown of NPC1L1 activity, we sought to determine if they were protected from hepatic triglyceride accumulation relative to individuals with wild-type NPC1L1.

The study was conducted in the Dallas Heart Study (DHS), a multiethnic population-based probability sample of Dallas County (Texas) weighted to include 50% black and 50% nonblack individuals (1043 whites, 1832 blacks, and 601 hispanics).1 Each participant completed a 60-minute structured questionnaire that provided detailed data regarding demographics, medication use, and ethanol intake. No participant used ezetimibe. The sequencing of DNA and assays for sequence variation in NPC1L1 were previously described8 as were the methods used to determine hepatic triglyceride content.10 The study was approved by the institutional review board (UT Southwestern), and all subjects provided written informed consent prior to participation.

A total of 128 DHS participants were found to be heterozygous for one of the following NS sequence variants in NPC1L1 associated with low intestinal absorption of cholesterol: T61M, N132S, R306C, D398G, R417W, G434R, T499M, S620C, I647N, R693C, S881L, W1014X, R1108W, L110F, R306C, A395V, G402S, T413M, I647N, G672R, R693C, R1214H, or R1268H.8 The study group was comprised 85 of these individuals (16 whites, 62 blacks, six hispanics, and one other) who had also undergone proton spectroscopy for determination of liver triglyceride content. The group included 42 women and 43 men.

To determine if NS sequence variations in NPC1L1 that confer a diminished capacity for intestinal cholesterol absorption were associated with low levels of hepatic triglycerides, we compared the liver fat content of heterozygotes for these variations with the levels in a group of DHS subjects with wild-type NPC1L1 who were matched for age, race/ethnicity, sex, and body mass index. The characteristics of these groups are presented in Table 1. The two groups demonstrated no differences in serum lipid profiles, glucose concentrations, insulin sensitivity, aminotransferases, or ethanol intake. The campesterol-to-lathosterol ratio, an indicator of dietary cholesterol absorption, was significantly lower among heterozygotes for an NPC1L1 mutant allele. Individuals with wild-type NPC1L1 were also more likely to be on statin therapy. Hepatic triglyceride content was similar between the groups as a whole and in the subgroups of women, men, whites, blacks, and hispanics (data not shown). These findings were not different when individuals taking a statin were excluded from the analysis (normal versus NPC1L1+/−: 3.2% [1.9%-6.0%] versus 3.8% [2.5%-5.4%]; P = 0.788).

Table 1. Subject Characteristics
CharacteristicNormal (n = 85)NPC1L1 +/− (n = 85)P Value
  1. Values are median with interquartile range. Data analyzed by Wilcoxon's two-sample test and chi-square test. ALT, alanine aminotransferase; AST, aspartate aminotransferase; HOMA, homeostasis model assessment of insulin resistance.

Age (years)46 (38-56)47 (39-56)0.885
Body mass index (kg/m2)30 (26-36)30 (26-36)0.957
Total cholesterol (mg/dL)182 (152-207)176 (154-204)0.461
Triglycerides (mg/dL)92 (68-136)111 (74-135)0.134
Glucose (mg/dL)93 (82-104)93 (84-101)0.974
HOMA3.0 (1.8-4.6)3.6 (1.8-5.4)0.614
AST (IU/L)21 (17-27)23 (17-30)0.525
ALT (IU/L)20 (15-29)19 (15-31)0.825
Ethanol intake (g/day)0.3 (0.0-5.6)0.4 (0.0-8.4)0.356
Campesterol:lathosterol ratio2.1 (1.4-3.4)1.3 (0.9-2.1)0.005
Statin use (%)2160.038
Hepatic triglyceride content (%)3.6 (2.2-8.0)3.6 (2.2-5.4)0.412

Contrary to the data from small studies of ezetimibe in patients with NAFLD,3-5 our data suggest that diminished capacity for absorption of dietary cholesterol via NPC1L1 is not associated with protection from hepatic triglyceride accumulation. Prior reports in rodents have also suggested that pharmacologic attenuation or genetic abrogation of NPC1L1 alleviates insulin resistance7; however, our data do not support any changes in glucose homeostasis in these individuals despite a diminished cholesterol uptake over their entire lifetime. These results do not negate the possibility that acute treatment with ezetimibe may have a beneficial effect in NAFLD, as suggested by preliminary studies.3-5 Heterozygotes for NPC1L1 deficiency presumably have a 50% reduction in sterol uptake, and it remains possible that more complete blockade of sterol absorption is required to lower liver fat content. Larger controlled trials will be required to answer this question.

References

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    Cohen JC, Pertsemlidis A, Fahmi S, Esmail S, Vega GL, Grundy SM, et al. Multiple rare variants in NPC1L1 associated with reduced sterol absorption and plasma low-density lipoprotein levels. Proc Natl Acad Sci U S A 2006; 103: 1810-1815.
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    Szczepaniak LS, Nurenberg P, Leonard D, Browning JD, Reingold JS, Grundy S, et al. Magnetic resonance spectroscopy to measure hepatic triglyceride content: prevalence of hepatic steatosis in the general population. Am J Physiol Endocrinol Metab 2005; 288: E462-E468.

Ruben Ramirez M.D.* †, Jonathan C. Cohen Ph.D.* †, Helen H. Hobbs M.D.* † ‡, Jeffrey D. Browning M.D.* ‡ §, * Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, TX, † Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, Dallas, TX, ‡ Department of Molecular Genetics, The University of Texas Southwestern Medical Center, Dallas, TX, § Advanced Imaging Research Center, The University of Texas Southwestern Medical Center, Dallas, TX.