Decreased Npc1 Gene Dosage in Mice Is Associated With Weight Gain

Abstract

A recent genome-wide association study has determined that the Niemann-Pick C1 (NPC1) gene is associated with early-onset and morbid adult obesity. However, what effects of the nonsynonymous variation in NPC1 on protein function result in weight gain remains unknown. The NPC1 heterozygous mouse model (Npc1^+/−), which expresses one-half the normal amounts of functional Npc1 protein compared to the homozygous normal (Npc1^+/+) mouse, was used to determine whether decreased Npc1 gene dosage was associated with weight gain when fed either a low-fat (10% kcal fat) or high-fat (45% kcal fat) diet beginning at 4 weeks of age until 20 weeks of age. The results indicated that Npc1^+/− mice had significantly increased weight gain beginning at 13 weeks of age when fed a high-fat diet, but not when fed a low-fat diet, compared to the Npc1^+/+ mice fed the same diet. With respect to mice fed a high-fat diet, the Npc1^+/− mice continued to have significantly increased weight gain to 30 weeks of age. At this age, the Npc1^+/− mice were found to have increased liver and inguinal adipose weights compared to the Npc1^+/+ mice. Therefore, decreased Npc1 gene dosage resulting in decreased Npc1 protein function, promoted weight gain in mice fed a high-fat diet consistent with a gene–diet interaction.

A recent report has indicated that the Niemann-Pick C1 (NPC1) gene, encoding the NPC1 protein that regulates the transport of lipoprotein-derived cholesterol from late endosomes/lysosomes to other cellular compartments and responsible for maintaining intracellular cholesterol homeostasis, is associated with early-onset and morbid adult obesity (1). The authors performed a genome-wide association study on nearly 1,400 obese Europeans compared to a similar number of age-matched normal-weight controls. The results were then confirmed with 2,100 obese and 2,400 nonobese individuals. The nonsynonymous single-nucleotide polymorphism variant (rs1805081) results in the replacement of an arginine for histidine at amino acid 215 (H215R). However, this report did not address whether the NPC1 variant increased or decreased NPC1 protein function to promote weight gain.

To address this question, we have used the NPC1 mouse model (Npc1^NIH) considered to be a near perfect model of the human disorder (2–5). An important genetic difference is that although most human NPC1 mutations are point mutations resulting in variable NPC1 protein function, the mouse Npc1 mutation is represented by a deletion mutation with no detectable Npc1 protein expressed (4,6). As a result, the Npc1 heterozygous (Npc1^+/−) mouse expresses one-half the normal amounts of functional Npc1 protein. This study used male Npc1^+/+ and Npc1^+/− mice, fed either a low-fat or high-fat diet, to determine whether decreased Npc1 gene dosage is associated with weight gain.

METHODS AND PROCEDURES

Mice

A breeding pair of BALB/cJ Npc1^NIH heterozygous (Npc1^+/−) mice was initially obtained from the Jackson Laboratory (Bar Harbor, ME). These mice were bred to generate Npc1 normal (Npc1^+/+) mice and additional Npc1 heterozygous (Npc1^+/−) mice that were interbred to obtain Npc1^+/+ and Npc1^+/− mice for this study. The mice were maintained at the University of Arizona Animal Care Facility.

Genotype analysis

At the time of weaning (21 days), the Npc1 mice were genetically identified using a PCR at the Npc1 locus as previously described (4). In brief, PCR amplification buffer was used with 20–40 ng of tail-tip DNA added at 85 °C, followed by amplification performed for 35 cycles of 30 s at 95 °C, 30 s at 61 °C, and 1 min at 72 °C, with a terminal extension for 10 min at 72 °C. The PCR products were separated using a 1.2% agarose gel and visualized with a UV light.

Study protocol

One week after weaning (28 days of age), the male Npc1^+/+ and Npc1^+/− mice were weighed and placed on either a low-fat diet (10% kcal fat or 4% g fat, Diet-07021301) or high-fat diet (45% kcal fat or 24% g fat, Diet-07021302) produced by Research Diets (New Brunswick, NJ). A concerted effort was made to place Npc1^+/+ and Npc1^+/− mice (3 or 4 mice per cage) within the same cage. As previously reported, compared to Npc1^+/+ mice, the Npc1^+/− mice express one-half the normal amounts of functional Npc1 protein, thereby providing a model to investigate decreased Npc1 gene dosage in the absence of disease (6,7). The Npc1^+/+ and Npc1^+/− mice fed a low-fat diet were weighed every week from 4 to 20 weeks of age, while the Npc1^+/+ and Npc1^+/− mice fed a high-fat diet were weighed every week from 4 to 30 weeks of age. At 30 weeks of age, the Npc1^+/+ and Npc1^+/− mice fed a high-fat diet were killed and the liver weights and inguinal adipose weights were determined.

Statistics

Quantitative data are represented as the mean ± s.e.m. of 14–16 Npc1^+/+ mice and 14–15 Npc1^+/− mice within each group (low-fat or high-fat diet). Significant differences (P ≤ 0.05) between body weight, liver weight, inguinal adipose weight, or growth curves were determined using conventional or repetitive measures ANOVA.

RESULTS

The results indicated no significant difference (P = 0.9960) in the growth curves of Npc1^+/+ and Npc1^+/− mice when fed a low-fat diet from 4 to 20 weeks of age (Figure 1a). However, the growth curve of Npc1^+/− mice was significantly increased (P = 0.0048) compared to Npc1^+/+ mice when fed a high-fat diet from 4 to 20 weeks of age (Figure 1b). Within this period (13–20 weeks of age), the Npc1^+/− mice had a significantly increased (8–9%, P < 0.05) body weight compared to Npc1^+/+ mice. When the Npc1^+/+ and Npc1^+/− mice continued to be fed a high-fat diet from 20 to 30 weeks of age, the growth curve of Npc1^+/− mice remained significantly increased (P = 0.0179) compared to Npc1^+/+ mice. At 30 weeks of age, the Npc1^+/− mice had a significantly increased (12.8%, P = 0.0011) body weight compared to Npc1^+/+ mice (Figure 1c). Consistent with an increased body weight of Npc1^+/− mice at 30 weeks of age, these mice had a significantly increased liver weight (26.8%, P = 6.86 × 10⁻⁵) and inguinal adipose weight (13.5%, P = 0.0435) compared to Npc1^+/+ mice (Figure 1d,e).

Figure 1.

Body weights, liver weights, and inguinal adipose weights of male Npc1^+/+ and Npc1^+/− mice. (a) Growth curves for male Npc1^+/+ and Npc1^+/− mice from 4 to 20 weeks of age when fed a low-fat (10% kcal fat) diet. (b) Growth curves for male Npc1^+/+ and Npc1^+/− mice from 4 to 20 weeks of age when fed a high-fat (45% kcal fat) diet. (c) Body weights for male Npc1^+/+ and Npc1^+/− mice at 30 weeks of age when fed a high-fat (45% kcal fat) diet. (d) Liver weights for male Npc1^+/+ and Npc1^+/− mice at 30 weeks of age when fed a high-fat (45% kcal fat) diet. (e) Inguinal adipose weights for male Npc1^+/+ and Npc1^+/− mice at 30 weeks of age when fed a high-fat (45% kcal fat) diet. The groups were represented with 14–15 mice. An asterisk indicates a significant difference (P ≤ 0.05).

DISCUSSION

The results of this study indicate that decreased Npc1 gene dosage within the proper environment has a significant effect on weight gain using the NPC1 mouse model. Specifically, although the Npc1^+/− mice, which express one-half the normal amounts of functional Npc1 protein, had no significant difference in growth rate or weight gain when fed a low-fat diet, the Npc1^+/− mice were found to have a significantly increased growth rate and weight gain beginning at 13 weeks of age when fed a high-fat diet.

The mechanism responsible for decreased Npc1 gene dosage in promoting weight gain remains undefined. However, a number of studies suggest that the cholesterol-transport protein caveolin-1 and impaired feedback inhibition of the sterol regulatory element-binding protein (SREBP) pathway may be involved. First, studies have demonstrated that Npc1^+/− mice fed a basal diet, although not susceptible to increased weight gain, have a nearly fourfold increased expression of liver caveolin-1 and hepatic steatosis compared to both Npc1^+/+ and Npc1^−/− mice (7,8). Second, a recent study has indicated that increased expression of caveolin-1 in human adipose is associated with obesity and type 2 diabetes mellitus (9). Third, caveolin-1 deficient (Cav-1^−/−) mice have been found to be lean and resistant to diet-induced obesity (10). Fourth, mutations in caveolin-1 have recently been found to be associated with a form of generalized lipodystrophy (11). And fifth, although early studies suggested that Npc1^+/− mice do not possess partial metabolic abnormalities commonly associated with NPC1 disease, more recent studies have determined that aged Npc1^+/− mice and cats accumulate cholesterol within certain tissues (brain and liver) and that relatively young Npc1^+/− mice have impaired feedback inhibition of the SREBP pathway, characterized in part by increased expression and processing of the SREBP-1 and SREBP-2 proteins capable of inducing lipogenesis (7,12,13). Therefore, it will be of importance to determine whether nonsynonymous single-nucleotide polymorphisms in human NPC1 might also affect expression and function of caveolin-1, or impaired feedback inhibition of the SREBP pathway. Alternatively, altered fat absorption due to effects of Npc1 on the enteric nervous system, may provide an explanation (14).

In summary, although effects of the nonsynonymous polymorphism (H215R) associated with early-onset morbid human obesity remains unknown, results from this study indicate that decreased Npc1 gene dosage is associated with weight gain in mice fed a high-fat diet, consistent with a gene–diet interaction. Although speculative, decreased Npc1 gene dosage in the presence of a high-fat diet may be unable to maintain normal energy and metabolic homeostasis. The exact mechanism responsible for decreased NPC1 gene dosage in promoting weight gain will be the subject of future research.