The Niemann-Pick C1 Gene Is Downregulated in Livers of C57BL/6J Mice by Dietary Fatty Acids, but Not Dietary Cholesterol, through Feedback Inhibition of the SREBP Pathway

David Jelinek; Joseph J Castillo; Lisa M Richardson; Li Luo; Randall A Heidenreich; William S Garver

doi:10.3945/jn.112.162818

. 2012 Sep 18;142(11):1935–1942. doi: 10.3945/jn.112.162818

The Niemann-Pick C1 Gene Is Downregulated in Livers of C57BL/6J Mice by Dietary Fatty Acids, but Not Dietary Cholesterol, through Feedback Inhibition of the SREBP Pathway^¹^²^³

David Jelinek ⁴, Joseph J Castillo ⁴, Lisa M Richardson ⁴, Li Luo ⁵, Randall A Heidenreich ⁶, William S Garver ^4,^*

PMCID: PMC3497932 PMID: 22990467

Abstract

The Niemann-Pick C1 (NPC1) gene is associated with human obesity. Mouse models with decreased Npc1 gene dosage are susceptible to weight gain when fed a high-fat diet, but not a low-fat diet, consistent with an Npc1 gene-diet interaction. The objectives of this study were to define regulation of the Npc1 gene and to investigate the Npc1 gene-diet interaction responsible for weight gain. The experimental design involved feeding C57BL/6J male mice a low-fat diet (with 0.00, 0.10, or 1.00% cholesterol) or a high-fat diet (with 0.02% cholesterol) until 30 wk to determine regulation of the Npc1 gene in liver. The key results showed that the Npc1 gene was downregulated by dietary fatty acids (54%, P = 0.022), but not by dietary cholesterol, through feedback inhibition of the sterol regulatory element–binding protein (SREBP) pathway. However, the dietary fatty acids secondarily increased liver cholesterol, which also inhibits the SREBP pathway. Similarly, the Npc1 gene was downregulated in peritoneal fibroblasts isolated from C57BL/6J weanling male mice not exposed to the experimental diets and incubated in media supplemented with purified oleic acid (37%, P = 0.038) but not in media supplemented with purified cholesterol. These results are important because they suggest a novel mechanism for the interaction of fatty acids with the Npc1 gene to influence energy balance and to promote weight gain. Moreover, the responsiveness of the Npc1 gene to fatty acids is consistent with studies that suggest that the encoded NPC1 protein has a physiologic role in regulating both cholesterol and fatty acid metabolism.

Introduction

The Niemann-Pick C1 (NPC1)⁷ gene has been primarily investigated in relation to an autosomal-recessive lipid-storage disorder characterized by hepatosplenomegaly and neurodegeneration, with death typically occurring in the second decade of life (1, 2). The NPC1 gene is localized to human chromosome 18q11-12 and encodes a complex membrane-bound protein with extensive structural homology to members of the resistance-nodulation-division family of prokaryotic permeases (3, 4). The NPC1 protein has a number of structural motifs, including a luminal oriented N-terminal domain and 5 membrane-spanning helices representing a sterol-sensing domain, both of which independently bind cholesterol (5–7). The NPC1 protein is associated with a unique, late endosome-like compartment that transiently interacts with late endosomes/lysosomes and the trans-Golgi network (8, 9). Although expression of the NPC1 gene remains undefined, a number of studies have shown that the NPC1 protein has a central role in regulating the transport of lipoprotein-derived lipid (cholesterol and fatty acids) from late endosomes/lysosomes to other cellular compartments (10, 11).

A recent genome-wide association study has shown that the NPC1 gene is associated with childhood and adult obesity in European populations (12). The ability of the NPC1 gene nonsynonymous single nucleotide polymorphisms (rs1805081 and rs1805082 encoding H215R and I858V, respectively) used to perform this study was unknown to increase or decrease NPC1 protein function in relation to weight gain. To address this question, we performed growth studies by using the BALB/cJ Npc1 mouse model that possesses a deletion mutation (13, 14). The results from this study showed that compared with wild-type or Npc1 homozygous normal (Npc1^+/+) mice, the Npc1 heterozygous (Npc1^+/−) mice with decreased gene dosage were susceptible to weight gain when fed a high-fat diet but not when fed a low-fat diet, consistent with an Npc1 gene-diet interaction in relation to weight gain (15). These results were confirmed and extended by using BALB/cJ Npc1^+/− mice bred with wild-type C57BL/6J mice to produce hybrid (BALB/cJ and C57BL/6J) Npc1^+/− mice that, when fed a high-fat diet, exhibited weight gain with associated metabolic features (16). Further analyses of both the BALB/cJ and hybrid Npc1^+/− mice suggested that the Npc1 gene interacts with a high-fat diet and unknown modifying genes to promote weight gain (17).

With respect to in vivo physiologic regulation of the Npc1 gene, studies have suggested that Npc1 gene expression is constitutive and not regulated in wild-type mice (both BALB/cJ and C57BL/6J mouse strains) when fed a high-cholesterol diet (18, 19). However, subsequent studies performed in vitro using human fibroblasts, mouse fibroblasts, and porcine granulosa cells indicated that the NPC1 or Npc1 gene is downregulated at the transcriptional level by feedback inhibition of the sterol regulatory element–binding protein (SREBP) pathway (20, 21). These studies also indicated that the NPC1 or Npc1 gene promoter region in humans, mice, and pigs possesses both sterol regulatory element (SRE)-like sequences and classic SRE sequences and is therefore capable of interacting with all 3 forms of mature (m)-SREBP proteins to regulate transcription of the NPC1 or Npc1 gene.

The objectives of this study were to define regulation of the Npc1 gene and to investigate the Npc1 gene-diet interaction responsible for weight gain. The experimental design was to feed C57BL/6J male mice 1 of 4 experimental diets formulated with different amounts of cholesterol and fat until 30 wk to determine the regulation of the Npc1 gene in liver. To verify results, peritoneal fibroblasts isolated from C57BL/6J weanling male mice not exposed to the experimental diets were incubated in media supplemented with purified cholesterol or oleic acid.

Materials and Methods

Materials.

The diets were defined as follows: 1) a low-fat diet without cholesterol [4.0% fat and 0.00% cholesterol (LFD+0.00C); Diet-07021301], 2) a low-fat diet with 0.10% cholesterol [4.0% fat and 0.10% cholesterol (LFD+0.10C); Diet-07021306], 3) a low-fat diet with 1.00% cholesterol [4.0% fat and 1.00% cholesterol (LFD+1.00C); Diet-07021307], and 4) a high-fat diet with 0.02% cholesterol [24% fat and 0.02% cholesterol derived from lard (HFD+0.02C); Diet-07021302]. These energy-balanced diets (same total % energy) were formulated and produced by Research Diets. The composition and fatty acid profile of these diets are provided in Supplemental Tables 1–3. The SREBP-1 antibody (10007663) was purchased from Cayman Chemical. The SREBP-2 antibody (100-2215) was purchased from Novus Biologicals. The NPC1 antibody, generated against amino acids 1254–1273 (NKAKSCATEERYGTERER) of the human NPC1 protein, was custom made, affinity-purified, and purchased from Invitrogen. The β-actin antibody (A5316) was purchased from Sigma Chemical Company.

Mice.

Wild-type male C57BL/6J weanling mice were obtained from the Jackson Laboratory and maintained at the University of New Mexico Health Sciences Center Animal Resources Facility. To ensure the ethical treatment of mice in this study, the experimental protocol was approved by the Institutional Animal Care and Use Committee and performed in accordance with accepted guidelines published in the NIH Guide for the Care and Use of Laboratory Animals.

Experimental design.

The wild-type C57BL/6J mice used to perform these studies were selected because they are susceptible to obesity and insulin resistance when fed a high-fat diet (22, 23). The mice were housed in a room maintained at 23–24°C, 31–32% humidity, with an alternating 12-h light/dark cycle. They were fed 1 of 4 diets and consumed water ad libitum. The mice and food were weighed on a weekly basis to determine body weights and food intake from 5 to 25 wk. The mice were food-deprived and killed by using CO₂ asphyxiation at 30 wk. The Npc1 and Srebp genes were examined in the mouse liver because these genes are more highly expressed and regulated by the flow of lipoprotein-derived dietary lipids in the liver compared with other tissues (18, 24). The Npc1 and Srebp genes were also examined in fibroblasts derived from wild-type mice as a result of previous characterization (8, 24).

Concentration of liver lipids.

The concentration of liver lipids (cholesterol, cholesteryl esters, FFA, and TG) was calculated after determining the mass of each lipid and normalizing it to the amount of liver protein precipitated during organic extraction as previously described (25).

Concentration of plasma components.

The concentration of plasma glucose was determined by using the Infinity Glucose kit (Thermo Scientific). The concentration of insulin was determined by using the Mouse High Range Insulin ELISA kit (ALPCO Immunoassays). Calculations were performed by using the concentrations of plasma glucose and insulin to determine defined measures of insulin resistance, including the homeostasis model assessment–insulin resistance index (HOMA-IR), the homeostasis model assessment–β cell function percentile (HOMA-β%), and the quantitative insulin sensitivity check index (QUICKI) (26, 27). The concentrations of plasma cholesterol and TG were determined by using the Infinity Cholesterol and Infinity Triacylglycerol kits (Thermo Scientific). The concentration of plasma FFA was determined by using the EnzyChrom Free Fatty Acid kit (BioAssay Systems). These assays were performed in the presence of normal control serum (Data-Trol N) and abnormal control serum (Data-Trol A; Thermo Scientific).

Mouse fibroblasts.

Mouse fibroblasts were derived from the peritoneal membrane of wild-type C57BL/6J weanling male mice as previously described (28). Note that these weanling male mice (21 d) were obtained from mice fed only a basal diet and not exposed to 1 of the 4 experimental diets. In brief, at ∼50% confluence (4th passage) the fibroblasts were rinsed with PBS and incubated in media with reduced FBS (DMEM, 5% FBS, 1% Penicillin/Streptomycin) or media with reduced lipid-deficient–FBS [DMEM, 5% lipid-deficient FBS (LD-FBS), 1% Penicillin/Streptomycin], the latter of which depletes cellular sterol and fatty acid pools and promotes physiologic upregulation of the SREBP pathway. At ∼90% confluence, the fibroblasts were rinsed with PBS and incubated in media with reduced FBS or LD-FBS supplemented with the following: 1) no lipids (control), 2) 130 μmol/L purified cholesterol, or 3) 45 μmol/L purified oleic acid. A dose-response study was also performed by incubating fibroblasts in media with reduced LD-FBS supplemented with increasing concentrations (0, 7, 14, 28, and 56 μmol/L) of purified oleic acid. The supplementation of purified lipids into media was accomplished by preparing stock solutions of cholesterol (25 mmol/L cholesterol in absolute ethanol) and oleic acid (35 mmol/L oleic acid in absolute ethanol) by using a volume no greater than 4.0 μL ethanol/1.0 mL media (0.4% v:v). The fibroblasts were incubated in media (37°C for 24 h), rinsed with ice-cold PBS, and scraped from plates, and total RNA was extracted to perform qRT-PCR.

RNA preparation and qRT-PCR.

Total RNA was extracted from mouse livers or mouse fibroblasts by using the RNeasy Mini Kit (Qiagen) and treated with RNase-free DNase to remove contaminating DNA. The relative amounts of mRNA transcribed from genes encoding acetyl-CoA carboxylase α (Acacα, 107476-Mm01304277_m1), fatty acid synthase (Fas, 14104-Mm00662319_m1), hydroxymethylglutaryl-CoA reductase (Hmgcr, 15357-Mm01282499_m1), LDL receptor (Ldlr, 16835-Mm00440169_m1), liver X receptor (Lxr, 22259-Mm00443454_m1), NPC1 (Npc1, 18145-Mm00435283_m1), SREBP-1 (Srebp-1, 20787- Mm00550338_m1 and 20788-Mm01138338_g1), and SREBP-2 (Srebp-2, 20788-Mm01306292_m1) were determined by using qRT-PCR. Reverse transcription was performed by using 2.5 μmol/L random hexamers, 4.0 mmol/L deoxyribonucleotide triphosphates, 15 mmol/L MgCl₂, 50 U reverse transcriptase, and 100 U RNA inhibitor to produce cDNA, followed by qRT-PCR using a TaqMan Gene Expression Assay containing predeveloped PCR primers and Mouse Genome Database probes (FAM dye-labeled) with an ABI-PRISM Sequence Detection System (Applied Biosystems). The relative amounts of target mRNA were normalized to 18S rRNA (internal control).

Western blot analysis.

The relative amounts of m-SREBP-1, m-SREBP-2, NPC1 protein, and β-actin (internal control) were determined by using Western blot analysis as previously described (25).

Npc1 gene promoter region analysis.

The nucleotide sequence for the mouse Npc1 gene promoter region was generously provided by Bruce D. Murphy (University of Montreal, Canada) and confirmed by using the nucleotide sequence obtained from the Mouse Genome Database Project (29, 30). The nucleotide sequence from 5000 bp upstream of the Npc1 gene translational start site was analyzed for consensus sequences previously shown to represent E-boxes (5′-ATCACGTGAT-3′), classic SREs (5′-ATCACcCCAC-3′), and SRE-like (5′-ATCGCcTGAT-3′) sequences by using the Mouse NCBI BLASTN algorithm (31).

Statistical analysis.

Kruskal-Wallis 1-way ANOVA was used to evaluate the overall differences between multiple groups of mice and fibroblasts. Pairwise comparisons between the control group and each treatment group were performed by using the Wilcoxon rank sum test. This test is a nonparametric alternative of the 2-sample t test used to perform repeated comparisons and all group comparisons. The false discovery rate method correcting for multiple comparisons was used to determine if the post hoc test was significant. Two-way repeated-measures ANOVA and the Bonferroni post hoc tests were used to assess the effects of diet, time, and diet × time interactions for body weight and cumulative energy intake. Data are expressed as means ± SE, and significance was considered at P < 0.05. All statistical analyses were performed by using statistical software SAS 9.3 (SAS Institute).

Results

Body weights, liver weights, concentration of liver lipids, and concentration of plasma components in mice fed experimental diets.

The mice fed HFD+0.02C had a significantly greater body weight than all of the groups fed a low-fat diet (Table 1). These differences in body weight were not attributable to differences in energy intake as indicated by the cumulative energy intake normalized to body weight (Supplemental Fig. 1). Moreover, mice fed HFD+0.02C had a significantly greater liver weight than all of the groups fed a low-fat diet, although mice fed LFD+1.00C had a significantly greater liver weight than mice fed LFD+0.00C. The mice fed LFD+1.00C had a significantly greater concentration of liver cholesterol than mice fed LFD+0.00C or LFD+0.10C. However, mice fed HFD+0.02C had a significantly greater concentration of liver cholesterol than all of the groups fed a low-fat diet. In contrast to the concentration of liver cholesterol, mice fed LFD+0.10C, LFD+1.00C, or HFD+0.02C had significantly greater concentrations of liver cholesteryl esters than mice fed LFD+0.00C. The mice fed LFD+0.10C, LFD+1.00C, or HFD+0.02 had significantly greater liver FFA concentrations than mice fed LFD+0.00C. The mice fed HFD+0.02C had a significantly greater concentration of liver TG than all of the groups fed a low-fat diet. The mice fed HFD+0.02C had significantly greater concentrations of plasma glucose and insulin than all of the groups fed a low-fat diet. Consistent with these results, mice fed HFD+0.02C had insulin resistance, as indicated by significantly greater HOMA-IR and HOMA-β% values, but a significantly lower QUICKI value than mice fed LFD+0.00C or LFD+0.10C. Although there were no significant differences in the concentration of plasma glucose and insulin between mice fed LFD+0.10C and LFD+1.00C, those fed LFD+1.00C had a significantly lower HOMA-IR value than mice fed LFD+0.00C or LFD+0.10C, suggesting that a high percentage of dietary cholesterol actually enhanced insulin sensitivity when using this measure of insulin resistance. The presence of insulin resistance among mice fed HFD+0.02C was confirmed by performing glucose and insulin tolerance tests, whereby mice fed this diet had significantly greater glucose and insulin intolerance than all of the groups fed a low-fat diet (Supplemental Fig. 2). Finally, there were no significant differences in the concentration of plasma total TG between mice fed any of the 4 diets. Moreover, there were no significant differences in the concentration of plasma total cholesterol between mice fed a low-fat diet, although the concentration of plasma total cholesterol was significantly greater in mice fed HFD+0.02C.

TABLE 1.

Effect of dietary cholesterol and fat on body weights, liver weights, concentration of liver lipids, and concentration of plasma components in mice fed experimental diets until 30 wk¹

	LFD+0.00C	LFD+0.10C	LFD+1.00C	HFD+0.02C
Body weight, g	34.3 ± 3.7^b	37.5 ± 3.3^b	35.3 ± 3.6^b	46.7 ± 3.3^a
Liver weight, g	1.4 ± 0.3^c	1.6 ± 0.2^cb	1.8 ± 0.5^b	2.7 ± 0.7^a
C, nmol/mg protein	19.4 ± 1.3^c	20.4 ± 3.1^c	34.1 ± 2.1^b	61.3 ± 5.2^a
CE, nmol/mg protein	5.1 ± 2.4^c	31.0 ± 2.6^b	86.8 ± 10.9^a	33.3 ± 2.6^b
FFA, nmol/mg protein	80.4 ± 3.5^b	111 ± 6.7^a	142 ± 19.1^a	110 ± 7^a
TG, nmol/mg protein	0.83 ± 0.14^b	0.83 ± 0.18^b	0.75 ± 0.08^b	1.81 ± 0.27^a
Glucose, mmol/L	8.5 ± 0.4^b	8.1 ± 0.3^b	7.9 ± 0.3^b	10.6 ± 0.4^a
Insulin, nmol/L	0.18 ± 0.02^b	0.17 ± 0.02^b	0.13 ± 0.01^b	0.79 ± 0.09^a
HOMA-IR	0.31 ± 0.05^a	0.30 ± 0.03^a	0.22 ± 0.05^b	1.72 ± 0.22^c
HOMA-β, %	3.36 ± 0.04^b	3.68 ± 0.03^b	3.08 ± 0.04^b	10.2 ± 1.1^a
QUICKI	0.48 ± 0.01^a	0.49 ± 0.01^a	0.52 ± 0.01^a	0.36 ± 0.01^b
Plasma TTG, mmol/L	0.81 ± 0.04	0.88 ± 0.04	0.80 ± 0.05	0.82 ± 0.03
Plasma TC, mmol/L	3.75 ± 0.21^b	3.94 ± 0.10^b	3.87 ± 0.14^b	5.91 ± 0.32^a

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Values are means ± SE; n = 9–11. Means without a common letter differ, P < 0.05. C, cholesterol; CE, cholesteryl ester; HFD+0.02C, high-fat diet with 0.02% cholesterol; HOMA-β%, homeostasis model assessment–β cell function percentile; HOMA-IR, homeostasis model assessment–insulin resistance index; LFD+0.00C, low-fat diet with 0.00% cholesterol; LFD+0.10C, low-fat diet with 0.10% cholesterol; LFD+1.00C, low-fat diet with 1.00% cholesterol; QUICKI, quantitative insulin sensitivity check index; TC, total cholesterol; TTG, total triglyceride.

Relative amounts of liver Srebp and target gene mRNA for mice fed experimental diets.

The mice fed HFD+0.02C had significantly less liver Srebp-1 mRNA than mice fed LFD+0.00C (Table 2). With regard to the alternative transcripts of Srebp-1 mRNA being measured, a previous study performed using C57BL/6J mice fed a low-fat diet without the addition of cholesterol showed that the relative amounts of liver Srebp-1a mRNA to Srebp-1c mRNA are present at a ratio of 1 to 8.8 (24). The Srebp-1c mRNA alternatively transcribed from the Srebp-1 gene in mouse liver encodes a precursor (p)-SREBP-1c protein that eventually serves as a transcription factor for target genes, such as the Acacα and Fas genes, which are responsible for regulating the synthesis of FFA and TG, respectively. The mice fed LFD+1.00C and HFD+0.02C had significantly less liver Acacα mRNA than mice fed LFD+0.00C or LFD+0.10C, whereas mice fed LFD+0.10C, LFD+1.00C, or HFD+0.02C had significantly less liver Fas mRNA than mice fed LFD+0.00C. In contrast to the relative amounts of Srebp-1 mRNA, mice fed LFD+0.10C or LFD+1.00C had significantly less liver Srebp-2 mRNA than mice fed LFD+0.00C. The mice fed HFD+0.02C had marginally less liver Srebp-2 mRNA than mice fed LFD+0.00C (0.05 < P ≤ 0.10) but significantly more liver Srebp-2 mRNA than mice fed LFD+1.00C. The Srebp-2 mRNA transcribed from the Srebp-2 gene in mouse liver encodes the p-SREBP-2 protein that eventually serves as a transcription factor for target genes, such as the Ldlr and Hmgcr genes, which are responsible for regulating the endocytosis of lipoproteins and synthesis of cholesterol, respectively. The mice fed LFD+1.00C had significantly less liver Ldlr and Hmgcr mRNA than mice fed LFD+0.00C. There were no significant differences in the amounts of liver Ldlr and Hmgcr mRNA for mice fed LFD+0.00C and HFD+0.02C. The mice fed HFD+0.02C had 56% less liver Npc1 mRNA than mice fed LFD+0.00C, but the post hoc analysis with the 4 groups of mice indicated no significant difference (P = 0.06).

TABLE 2.

Effect of dietary cholesterol and fat on liver Srebp and target gene mRNA in mice fed experimental diets until 30 wk¹

Target mRNA	LFD+0.00C	LFD+0.10C	LFD+1.00C	HFD+0.02C
	Fold of LFD+0.00C
Srebp-1	1.00 ± 0.16^a	0.62 ± 0.06^a,b	0.64 ± 0.08^a,b	0.44 ± 0.04^b
Acacα	1.00 ± 0.25^a	1.13 ± 0.50^a	0.14 ± 0.07^b	0.03 ± 0.01^b
Fas	1.00 ± 0.28^a	0.23 ± 0.07^b	0.22 ± 0.03^b	0.29 ± 0.08^b
Srebp-2	1.00 ± 0.10^a	0.55 ± 0.08^b,c	0.42 ± 0.04^b	0.68 ± 0.04^a,c
Ldlr	1.00 ± 0.24^a	0.40 ± 0.09^a	0.35 ± 0.06^b	0.59 ± 0.10^a
Hmgcr	1.00 ± 0.31^a	0.23 ± 0.04^b	0.19 ± 0.06^b	0.34 ± 0.05^a
Npc1	1.00 ± 0.16	0.98 ± 0.06	0.85 ± 0.08	0.44 ± 0.04

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Values are means ± SE; n = 8. Means without a common letter differ, P < 0.05. Acacα, acetyl-CoA carboxylase α Fas, fatty acid synthase; HFD+0.02C, high-fat diet with 0.02% cholesterol; Hmgcr, hydroxymethylglutaryl-CoA reductase; Ldlr, LDL receptor; LFD+0.00C, low-fat diet with 0.00% cholesterol; LFD+0.10C, low-fat diet with 0.10% cholesterol; LFD+1.00C, low-fat diet with 1.00% cholesterol; Npc1, Niemann-Pick C1; Srebp, sterol regulatory element–binding protein.

Relative amounts of liver m-SREBP-1, m-SREBP-2, and NPC1 protein in mice fed experimental diets.

The mice fed LFD+1.00C and HFD+0.02C had significantly less liver m-SREBP-1 protein than mice fed LFD+0.00C or LFD+0.10C (Fig. 1). Moreover, mice fed LFD+1.00C had significantly less liver m-SREBP-1 protein than mice fed HFD+0.02C. There were no significant differences in liver m-SREBP-2 between mice fed any of the 4 diets. The mice fed LFD+0.10C or LFD+1.00C had significantly more liver NPC1 protein than mice fed LFD+0.00C and HFD+0.02C. In direct contrast, the mice fed HFD+0.02C had significantly less liver NPC1 protein than mice fed LFD+0.00C. Representative Western blots showing the relative amounts of liver m-SREBP-1, m-SREBP-2, NPC1 protein, and β-actin (internal control) are provided in Supplemental Fig. 3.

Effects of feeding C57BL/6J male mice 1 of 4 experimental diets until 30 wk on the relative amounts of liver m-SREBP-1, m-SREBP-2, and NPC1 protein. Values are means ± SE; n = 8. Means without a common letter differ, P < 0.05. HFD+0.02C, high-fat diet with 0.02% cholesterol; LFD+0.00C, low-fat diet with 0.00% cholesterol; LFD+0.10C, low-fat diet with 0.10% cholesterol; LFD+1.00C, low-fat diet with 1.00% cholesterol; m, mature; NPC1, Niemann-Pick C1; SREBP, sterol regulatory element–binding protein.

Relative amounts of fibroblast Srebp-1, Srebp-2, Lxr, and Npc1 mRNA when incubated in media supplemented with purified cholesterol or oleic acid.

The fibroblasts incubated in media with reduced FBS supplemented with cholesterol or oleic acid showed no significant differences in Srebp-1 mRNA than fibroblasts incubated in the same media without lipids (Fig. 2). However, fibroblasts incubated in media with reduced LD-FBS (used to deplete cellular sterol and fatty acid pools and promote physiologic upregulation of the SREBP pathway) supplemented with oleic acid had significantly less Srebp-1 mRNA than fibroblasts incubated in the same media without or with cholesterol. With regard to the alternative transcripts of Srebp-1 mRNA being measured, a previous study performed using C57BL/6J mouse fibroblasts incubated in basic media with FBS showed that the relative amounts of Srebp-1a mRNA to Srebp-1c mRNA are present at a ratio of 1 to 0.5 (24). The fibroblasts incubated in media with reduced LD-FBS, but not reduced FBS, supplemented with cholesterol or oleic acid had significantly less Srebp-2 mRNA than fibroblasts incubated in the same media without lipids. The fibroblasts incubated in media with reduced FBS or LD-FBS supplemented with cholesterol or oleic acid showed no significant differences in Lxr mRNA than fibroblasts incubated in the same media without lipids. Similar to the amounts of Srebp-1 mRNA, fibroblasts incubated in media with reduced LD-FBS supplemented with oleic acid had significantly less Npc1 mRNA than fibroblasts incubated in the same media without or with cholesterol. To confirm these results, a dose-response study was performed in media with reduced LD-FBS supplemented with increasing concentrations of oleic acid. The fibroblasts incubated in media with reduced LD-FBS supplemented with lower concentrations of oleic acid (7 and 14 μmol/L) showed no significant differences in Srebp-1, Srebp-2, and Npc1 mRNA. However, fibroblasts incubated in the same media supplemented with higher concentrations of oleic acid (28 and 56 μmol/L) showed significantly less Srebp-1, Srebp-2, and Npc1 mRNA than fibroblasts incubated in the same media without oleic acid.

Effects of incubating C57BL/6J weanling peritoneal mouse fibroblasts in media supplemented with purified cholesterol or oleic acid for 24 h on the relative amounts of *Srebp-1* mRNA (A), *Srebp-2* mRNA (B), *Lxr* mRNA (C), or *Npc1* mRNA (D) and in media supplemented with increasing concentrations of purified oleic acid for 24 h on the relative amounts of *Srebp-1*, *Srebp-2*, and *Npc1* mRNA (E). Values are means ± SE; n = 6 plates of fibroblasts. Means without a common letter differ, P < 0.05. Cholesterol, media supplemented with 130 μmol/L cholesterol; Control, media without supplemented lipid; LD-FBS, lipid-deficient FBS; Oleic acid, media supplemented with 45 μmol/L oleic acid; NPC1, Niemann-Pick C1; SREBP, sterol regulatory element–binding protein.

Mouse Npc1 gene promoter analysis.

The mouse Npc1 gene promoter region was analyzed to identify the presence of E-boxes and SRE capable of binding m-SREBP-1a, m-SREBP-1c, and m-SREBP-2 proteins. The results indicated an absence of nucleotide sequences having homology to E-boxes, even at 5000 bp upstream from the translational start site of the Npc1 gene. However, 2 different SRE with high homology to the prescribed mouse consensus sequences were located within 500 bp from the translational start site of the Npc1 gene as provided (Supplemental Fig. 4). An SRE-like sequence (5′-TCGCCTG-3′) was positioned in the forward orientation at −418 to −412 bp, whereas a classic SRE sequence (5′-GGGGTGA-3′) was positioned in the reverse orientation at −393 to −387 bp. The only other SRE found within close proximity from the translation start site of the Npc1 gene was another SRE-like sequence (5′-CAGGCGA-3′) positioned in the reverse orientation at −1477 to −1471 bp (data not shown).

Discussion

We previously showed that the Npc1 gene is downregulated by feedback inhibition of the SREBP pathway and that Npc1 mouse models with decreased gene dosage are susceptible to weight gain when fed a high-fat diet, but not when fed a low-fat diet, which is consistent with an Npc1 gene-diet interaction (15–17, 20). The objectives of this study were to define regulation of the Npc1 gene and to investigate the Npc1 gene-diet interaction responsible for weight gain. The experimental design was to feed C57BL/6J male mice a low-fat diet (with 0.00, 0.10, or 1.00% cholesterol) or a high-fat diet (with 0.02% cholesterol) until 30 wk to determine the regulation of the Npc1 gene in liver. The key results showed that the Npc1 gene is downregulated by dietary fatty acids (54%, P = 0.022), but not dietary cholesterol, through feedback inhibition of the SREBP pathway. However, the dietary fatty acids secondarily increased liver cholesterol, which also inhibits the SREBP pathway. Similarly, the Npc1 gene was downregulated in peritoneal fibroblasts isolated from C57BL/6J weanling male mice not exposed to the experimental diets and incubated in media supplemented with purified oleic acid (37%, P = 0.038) but not in media supplemented with purified cholesterol. These results are important because they suggest a novel mechanism for fatty acids interacting with the Npc1 gene to influence energy balance and to promote weight gain. Moreover, the responsiveness of the Npc1 gene to fatty acids is consistent with studies suggesting that the encoded NPC1 protein has a physiologic role in regulating both cholesterol and fatty acid metabolism.

In this study, the concentration of sterol (cholesterol and cholesteryl ester) and FFA were significantly greater in the livers of mice fed LFD+0.10C, LFD+1.00C, or HFD+0.02C than of mice fed LFD+0.00C (control diet). It is important to note that the concentration of cholesterol in the livers of mice fed HFD+0.02C (with only 0.02% cholesterol) was markedly greater (79%) than of mice fed LFD+1.00C (1.00% cholesterol). The metabolic basis for this particular result has been defined by SFA, which are present at increased amounts in the high-fat diet, inhibiting acyl-CoA:cholesterol acyltransferase and therefore preventing proper storage of excess intracellular cholesterol within lipid bodies (32, 33). As a result, this excess intracellular cholesterol becomes available to promote transcriptional and post-translational feedback inhibition of the SREBP pathway (34). Consistent with these studies, the livers of mice fed LFD+1.00C or HFD+0.02C had significantly less Srebp-1 and Srebp-2 mRNA, respectively, and target gene mRNA than mice fed LFD+0.00C.

With respect to the increased concentration of FFA in livers of these mice, previous studies have shown that unsaturated fatty acids, including oleic acid, antagonize the liver X receptor, which serves as a transcription factor for the Srebp-1 gene (35, 36). Moreover, in addition to sterols (cholesterol and oxysterol) inhibiting the SREBP cleavage activating protein and insulin-induced gene (INSIG) protein, respectively, studies indicate that unsaturated fatty acids inhibit the UBXD8 protein (a fatty acid–sensing protein that prevents degradation of INSIG) to potentiate feedback inhibition at the post-translational level, which decreases subsequent production of liver m-SREBP-1c protein (37, 38). The decreased m-SREBP-1c protein is believed to serve as the primary mechanism for decreasing transcription of the Srebp-1 gene through what is now referred to as an “autoloop regulatory circuit” (39, 40). A similar mechanism had previously been proposed for the SREBP-2 pathway, whereby feedback inhibition at the post-translational level and decreased m-SREBP-2 protein serve to decrease transcription of the Srebp-2 gene (41). An unexpected finding in the present study was that livers from mice fed LFD+0.10C or LFD+1.00C had significantly greater amounts of NPC1 protein, independent of transcriptional regulation by the m-SREBP-1 and -2 proteins, as evidenced by similar amounts of Npc1 mRNA. Although speculative, this particular result is believed to be due to inhibited ubiquitylation of the NPC1 protein (through the sterol-sensing domain binding of cholesterol) and targeting of the modified NPC1 protein to proteosomes for degradation as previously reported (42). Together, the livers of mice fed HFD+0.02C enriched with fatty acids, but not LFD+1.00C enriched with cholesterol, and mouse fibroblasts incubated in media with reduced LD-FBS supplemented with oleic acid, but not LD-FBS supplemented with cholesterol, were associated with significantly less Npc1 mRNA and NPC1 protein.

The ability of fatty acids to decrease transcription of the Npc1 gene was supported by the presence of specific SRE consensus sequences present in the Npc1 gene promoter region. Nucleotide sequence analysis of the mouse Npc1 gene promoter region revealed the presence of a forward SRE-like sequence (5′-TCGCCTG-3′ present at −418 to −412 bp) and a reverse classic SRE sequence (5′-GGGGTGA-3′ present at −393 to −387 bp) as indicated by using the NCBI database. In brief, m-SREBP-1c and m-SREBP-2 proteins have been reported to promote moderate transcriptional activity through binding of the SRE-like sequence, whereas m-SREBP-1a protein (present at less amounts in mouse liver) promotes high transcriptional activity through binding of this same sequence (31). Because the relative amounts of m-SREBP-1c protein, but not m-SREBP-2 protein, were significantly less in livers of mice fed HFD+0.02C, the m-SREBP-1c protein is proposed to have a principal role in downregulating transcription of the Npc1 gene. In contrast, m-SREBP-1a and m-SREBP-2 proteins have been reported to promote high transcriptional activity through binding of the classic SRE sequence, which therefore suggests that both proteins may also have a role in downregulating transcription of the Npc1 gene (31). In support of these results, previous studies performed in vitro have shown that both m-SREBP-1a and m-SREBP-1c proteins have high transcriptional activity and m-SREBP-2 protein has moderate transcriptional activity for regulating expression of the NPC1 gene in human cells and Npc1 gene in mouse cells (20, 21). A schematic representation has been provided outlining feedback inhibition of the SREBP pathway and transcriptional activity of the SREBP proteins in relation to downregulation of the Npc1 gene as suggested in this study (Fig. 3).

Schematic representation outlining feedback inhibition of the SREBP pathway (A) and transcriptional activity of the SREBP proteins in relation to downregulation of the *Npc1* gene (B). The SREBP-1 pathway is feedback inhibited at the transcriptional level by fatty acids inhibiting LXR and decreasing the amounts of *Srebp-1* mRNA. Alternative splicing of *Srebp-1* mRNA is responsible for the relative amounts of *Srebp-1a* and *Srebp-1c* mRNA. It has been reported that the relative amounts of *Srebp-1a* mRNA to *Srebp-1c* mRNA are present at a ratio of 1 to 8.8 in mouse liver and at a ratio of 1 to 0.5 in mouse fibroblasts. The SREBP-1 pathway is also feedback inhibited at the post-translational level by fatty acids that inhibit UBXD8 and sterols (cholesterol and oxysterol) that inhibit SCAP and INSIG, respectively, to decrease the amounts of m-SREBP-1a and m-SREBP-1c protein. In contrast, the SREBP-2 pathway is feedback inhibited at both the transcriptional and post-translational levels by sterol to decrease the amounts of *Srebp*-2 mRNA and m-SREBP-2 protein. The m-SREBP-1a protein has high transcriptional activity for both SRE-like and classic SRE sequences (*bold lines/arrows*), whereas the m-SREBP-1c protein has moderate transcriptional activity for the SRE-like sequence (*medium line/arrow*). Similarly, the m-SREBP-2 protein has moderate transcriptional activity for the SRE-like sequence (*medium line/arrow*) and high transcriptional activity for the classic SRE sequence (*bold line/arrow*). m, mature; p, precursor; SRE, sterol regulatory element; SREBP, sterol regulatory element–binding protein.

In summary, receptor-mediated endocytosis of lipoproteins enriched with phospholipids, cholesteryl esters, and TG results in the accumulation of constituent lipids (cholesterol and FFA) within the lumen of late endosomes/lysosomes. It is now known that the NPC1 protein has a central role in regulating the transport of cholesterol and FFA from late endosomes/lysosomes to other cellular compartments to maintain cellular, tissue, and whole-body lipid homeostasis. The key results showed that the Npc1 gene is downregulated by dietary or media fatty acids, but not cholesterol, through feedback inhibition of the SREBP pathway. However, the dietary fatty acids secondarily increased liver cholesterol, which also inhibits this pathway. These results are important because they suggest at least one mechanism for dietary fatty acids interacting with the Npc1 gene to influence energy balance and to promote weight gain. The responsiveness of the Npc1 gene to dietary fatty acids and secondarily to altered cellular cholesterol homeostasis is consistent with studies that suggest that the encoded NPC1 protein has a physiologic role in regulating both cholesterol and fatty acid metabolism.

Supplementary Material

Online Supporting Material

supp_142_11_1935__index.html^{(968B, html)}

Acknowledgments

The authors express appreciation to Dr. Renee C. LeBoeuf for providing valuable technical insight received through the Seattle Mouse Metabolic Phenotyping Center at the University of Washington. D.J., R.A.H., and W.S.G. designed research; D.J., J.J.C., L.M.R., and W.S.G. conducted research; D.J., L.L., and W.S.G. analyzed data; D.J., L.L., R.A.H., and W.S.G. wrote the manuscript; and W.S.G. had primary responsibility for final content. All authors read and approved the final manuscript.

Footnotes

Supported in part by the National Center for Research Resources and the National Center for Advancing Translational Sciences by National Institutes of Health grants UL1 TR000041 (to L.L.), R21 DK071544, P30 DK017047, a grant from the Tohono O’odham Nation, and private donations for the investigation of childhood genetic and metabolic diseases (to W.S.G.).

Supplemental Tables 1–3 and Supplemental Figures 1–4 are available from the “Online Supporting Material” link in the online posting of the article and from the same link in the online table of contents at http://jn.nutrition.org.

⁷

Abbreviations used: HFD+0.02C, high-fat diet with 0.02% cholesterol;HOMA-b%, homeostasis model assessment-b cell function percentile; LD-FBS,lipid-deficient FBS; LFD+0.00C, low-fat diet with 0.00% cholesterol; LFD+0.10C,low-fat diet with 0.10% cholesterol; LFD+1.00C, low-fat diet with 1.00%cholesterol; m, mature; NPC1, Niemann-Pick C1; QUICKI, quantitative insulinsensitivity check index; SRE, sterol regulatory element; SREBP, sterol regulatoryelement-binding protein.

Literature Cited

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Associated Data

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Supplementary Materials

Online Supporting Material

supp_142_11_1935__index.html^{(968B, html)}

supp_jn.112.162818_162818-STable1-STable3.pdf^{(1.5MB, pdf)}

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[bib1] 1.Garver WS, Francis GA, Jelinek D, Shepherd G, Flynn J, Castro G, Walsh Vockley C, Coppock DL, Pettit KM, Heidenreich RA, et al. The National Niemann-Pick C1 disease database: report of clinical features and health problems. Am J Med Genet. 2007;143A:1204–11 [DOI] [PubMed] [Google Scholar]

[bib2] 2.Imrie J, Dasgupta S, Besley GTN, Harris C, Heptinstall L, Knight S, Vanier MT, Fensom AH, Ward C, Jacklin E, et al. The natural history of Niemann-Pick disease type C in the UK. J Inherit Metab Dis. 2007;30:51–9 [DOI] [PubMed] [Google Scholar]

[bib3] 3.Carstea ED, Morris JA, Coleman KG, Loftus SK, Zhang D, Cummings C, Gu J, Rosenfeld MA, Pavan WJ, Kritzman DB, et al. Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis. Science. 1997;277:228–31 [DOI] [PubMed] [Google Scholar]

[bib4] 4.Davies JP, Chen FW, Ioannou YA. Transmembrane molecular pump activity of Niemann-Pick C1 protein. Science. 2000;290:2295–8 [DOI] [PubMed] [Google Scholar]

[bib5] 5.Davies JP, Ioannou YA. Topological analysis of Niemann-Pick C1 protein reveals that the membrane orientation of the putative sterol-sensing domain is identical to those of 3-hydroxy-3-methylglutaryl-CoA reductase and sterol regulatory element-binding protein cleavage-activating protein. J Biol Chem. 2000;275:24367–74 [DOI] [PubMed] [Google Scholar]

[bib6] 6.Infante RE, Radhakrishnan A, Abi-Mosleh L, Kinch LN, Wang ML, Grishin NV, Goldstein JL, Brown MS. Purified NPC1 protein: II. Localization of sterol binding to a 240-amino acid soluble luminal loop. J Biol Chem. 2008;283:1064–75 [DOI] [PubMed] [Google Scholar]

[bib7] 7.Ohgami N, Ko DC, Thomas M, Scott MP, Chang CCY, Chang TY. Binding between the Niemann-Pick C1 protein and a photoactivatable cholesterol analog requires a functional sterol-sensing domain. Proc Natl Acad Sci USA. 2004;101:12473–8 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib8] 8.Garver WS, Heidenreich RA, Erickson RP, Thomas MA, Wilson JM. Localization of the murine Niemann-Pick C1 protein to two distinct intracellular compartments. J Lipid Res. 2000;41:673–87 [PubMed] [Google Scholar]

[bib9] 9.Higgins ME, Davies JP, Chen FW, Ioannou YA. Niemann-Pick C1 is a late endosome-resident protein that transiently associates with lysosomes and the trans-Golgi network. Mol Genet Metab. 1999;68:1–13 [DOI] [PubMed] [Google Scholar]

[bib10] 10.Chen FW, Gordon RE, Ioannou YA. NPC1 late endosomes contain elevated levels of non-esterified ('free’) fatty acids and an abnormally glycosylated form of the NPC2 protein. Biochem J. 2005;390:549–61 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib11] 11.Liscum L, Ruggiero RM, Faust JR. The intracellular transport of low density lipoprotein-derived cholesterol is defective in Niemann-Pick type C fibroblasts. J Cell Biol. 1989;108:1625–36 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib12] 12.Meyre D, Delplanque J, Chevre JC, Lecoeur C, Lobbens S, Gallina S, Durand E, Vatin V, Degraeve F, Proenca C, et al. Genome-wide association study for early-onset and morbid adult obesity identifies three new risk loci in European populations. Nat Genet. 2009;41:157–9 [DOI] [PubMed] [Google Scholar]

[bib13] 13.Hsu SJ, Erickson RP, Zhang J, Garver WS, Heidenreich RA. Fine linkage and physical mapping suggests cross-over suppression with a retroposon insertion at the Npc1 mutation. Mamm Genome. 2000;11:774–8 [DOI] [PubMed] [Google Scholar]

[bib14] 14.Loftus SK, Morris JA, Carstea ED, Gu JZ, Cummings C, Brown A, Ellison J, Ohno K, Rosenfeld MA, Tagle DA, et al. Murine model of Niemann-Pick C disease: mutation in a cholesterol homeostasis gene. Science. 1997;277:232–5 [DOI] [PubMed] [Google Scholar]

[bib15] 15.Jelinek D, Heidenreich RA, Erickson RP, Garver WS. Decreased Npc1 gene dosage in mice is associated with weight gain. Obesity (Silver Spring). 2010;18:1457–9 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib16] 16.Jelinek D, Millward V, Birdi A, Trouard TP, Heidenreich RA, Garver WS. Npc1 haploinsufficiency promotes weight gain and metabolic features associated with insulin resistance. Hum Mol Genet. 2011;20:312–21 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib17] 17.Jelinek D, Heidenreich RA, Garver WS. The Niemann-Pick C1 gene interacts with a high-fat diet and modifying genes to promote weight gain. Am J Med Genet. 2011;155A:2317–9 [DOI] [PubMed] [Google Scholar]

[bib18] 18.Garver WS, Xie C, Repa JJ, Turley SD, Dietschy JM. Niemann-Pick C1 expression is not regulated by the amount of cholesterol flowing through cells in the mouse. J Lipid Res. 2005;46:1745–54 [DOI] [PubMed] [Google Scholar]

[bib19] 19.Maxwell KN, Soccio RE, Duncan EM, Sehayek E, Breslow JL. Novel putative SREBP and LXR target genes identified by microarray analysis in liver of cholesterol-fed mice. J Lipid Res. 2003;44:2109–19 [DOI] [PubMed] [Google Scholar]

[bib20] 20.Garver WS, Jelinek D, Francis GA, Murphy BD. The Niemann-Pick C1 gene is downregulated by feedback inhibition of the SREBP pathway in human fibroblasts. J Lipid Res. 2008;49:1090–102 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib21] 21.Gévry N, Schoonjans K, Guay F, Murphy BD. Cholesterol supply and SREBPs modulate transcription of the Niemann-Pick C-1 gene in steroidogenic tissues. J Lipid Res. 2008;49:1024–33 [DOI] [PubMed] [Google Scholar]

[bib22] 22.Alexander J, Chang GQ, Dourmashkin JT, Leibowitz SF. Distinct phenotypes of obesity-prone AKR/J, DBA2J and C57BL/6J mice compared to control strains. Int J Obes (Lond). 2006;30:50–9 [DOI] [PubMed] [Google Scholar]

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PERMALINK

The Niemann-Pick C1 Gene Is Downregulated in Livers of C57BL/6J Mice by Dietary Fatty Acids, but Not Dietary Cholesterol, through Feedback Inhibition of the SREBP Pathway123

David Jelinek

Joseph J Castillo

Lisa M Richardson

Li Luo

Randall A Heidenreich

William S Garver