Abstract
A genome-wide association study (GWAS) reported that common variation in the human Niemann-Pick C1 gene (NPC1) is associated with morbid adult obesity. This study was confirmed using our BALB/cJ Npc1 mouse model, whereby heterozygous mice (Npc1+/−) with decreased gene dosage were susceptible to weight gain when fed a high-fat diet (HFD) compared with homozygous normal mice (Npc1+/+) fed the same diet. The objective for our current study was to validate this Npc1 gene-diet interaction using statistical modeling with fitted growth trajectories, conduct body weight analyses for different measures, and define the physiological basis responsible for weight gain. Metabolic phenotype analysis indicated no significant difference between Npc1+/+ and Npc1+/− mice fed a HFD for food and water intake, oxygen consumption, carbon dioxide production, locomotor activity, adaptive thermogenesis, and intestinal lipid absorption. However, the livers from Npc1+/− mice had significantly increased amounts of mature sterol regulatory element-binding protein-1 (SREBP-1) and increased expression of SREBP-1 target genes that regulate glycolysis and lipogenesis with an accumulation of triacylglycerol and cholesterol. Moreover, white adipose tissue from Npc1+/− mice had significantly decreased amounts of phosphorylated hormone-sensitive lipase with decreased triacylglycerol lipolysis. Consistent with these results, cellular energy metabolism studies indicated that Npc1+/− fibroblasts had significantly increased glycolysis and lipogenesis, in addition to significantly decreased substrate (glucose and endogenous fatty acid) oxidative metabolism with an accumulation of triacylglycerol and cholesterol. In conclusion, these studies demonstrate that the Npc1 gene interacts with a HFD to promote weight gain through differential regulation of central energy metabolism pathways.
Keywords: adipose, fibroblast, liver, Niemann-Pick C1, obesity
the human niemann-pick C1 gene (NPC1) has been primarily investigated in relation to a rare autosomal-recessive lipid-storage disorder characterized by hepatosplenomegaly and progressive neurological degeneration that often results in death during the second decade (9, 17). The NPC1 gene is localized to chromosome 18 and encodes a complex membrane-bound protein that has extensive structural homology with members of the resistance-nodulation-division family of prokaryotic permeases (5, 6). With respect to the principal structural motifs, the NPC1 protein contains 13 membrane-spanning helices and 3 large luminal domains among which an NH2-terminal domain and sterol-sensing domain (SSD) independently bind cholesterol (18, 36). Although exact function of the NPC1 protein remains undefined, studies indicate that the NPC1 protein has a role in regulating the transport of lipid substrates (cholesterol, fatty acid, or sphingosine) across the limiting membrane of late endosomes into the cytoplasm (5, 29, 40). As a result, the characteristic phenotype for both cultured cells and tissues deficient in NPC1 protein function is an accumulation of these lipids in late endosomes and lysosomes (40, 45). A more recent study indicates that the NPC1 protein SSD forms a cavity in membrane bilayers and is capable of accommodating a cholesterol molecule consistent with this structural motif, serving as both a lipid-sensing and transport domain (26). With respect to expression of the NPC1 gene, it has been reported to be primarily regulated through the sterol regulatory element-binding protein (SREBP) pathway, consistent with the NPC1 protein having a primary role in maintaining cellular, tissue, and whole body lipid homeostasis (11, 13). Further studies have also indicated that the mouse Npc1 gene is regulated by fatty acids, but not cholesterol, through feedback inhibition of the SREBP-1 pathway (22).
A genome-wide association study (GWAS) has revealed that the human NPC1 gene is also associated with morbid adult obesity in European populations (32). This study was performed using nearly 1,400 obese Europeans compared with a similar number of age-matched normal weight controls and confirmed with an additional 2,100 obese and 2,400 normal weight individuals. When this study was published, it was unknown whether the NPC1 gene risk variants (644A>G encoding His215Arg and 2572A>G encoding Ile858Val) increased or decreased NPC1 protein function. To address this question and investigate the NPC1 gene in relation to weight gain, we performed growth studies using the BALB/cJ Npc1 mouse model, which possesses a retroposon insertion that prematurely terminates protein translation, thereby producing a nonfunctional truncated NPC1 protein (10, 15, 31). The results from this study demonstrated that compared with Npc1 homozygous normal (Npc1+/+) mice, the Npc1 heterozygous (Npc1+/−) mice with decreased gene dosage are susceptible to weight gain when fed a high-fat diet, but not when fed a low-fat diet (23). This study was later extended using BALB/cJ-C57BL/6J hybrid Npc1+/− mice that were also susceptible to weight gain and impaired glucose tolerance when fed a high-fat diet compared with hybrid Npc1+/+ mice fed the same diet (24). And more recently, we reported that C57BL/6J Npc1+/− mice are susceptible to weight gain when fed a high-fat diet compared with C57BL/6J Npc1+/+ mice fed the same diet (21). Moreover, an independent study has reported that rare human NPC1 gene loss-of-function mutations among male heterozygotes have a significantly higher BMI compared with matched controls or the whole population-based controls and that Npc1+/− mice fed a high-fat diet (HFD) have significantly increased fat storage compared with Npc1+/+ mice fed the same diet (28). Together, these results and an independent mouse GWAS using more than 100 strains of mice indicate that multiple Npc1 gene variants interact with a high-fat diet to increase body fat percentage and weight gain (39).
In general, it is believed that gene-environment interactions, in particular, gene-diet interactions, have an important role in propagating the worldwide epidemic of obesity (3, 37, 41). The concept of gene-diet interactions serve as a focal point for adaptation and evolutionary origins of obesity in different populations that may have occurred through neutral and positive selection (3, 41). To date ~130 human obesity genes have been identified, a subset of which are believed to interact with dietary components to promote weight gain (30). However, recent population-based studies to identify and quantify gene-diet interactions for obesity have resulted in low effect sizes due, in part, to limited validity of food questionnaires and ability to perform deep phenotype analysis (4, 33). Therefore, the objective for our current study was to validate this Npc1 gene-diet interaction using statistical modeling with fitted growth trajectories, conduct body weight analyses for different measures, and define the physiological basis responsible for weight gain.
MATERIALS AND METHODS
Mouse model.
A breeding pair of BALB/cNctr-Npc1m1N/J mice (hereafter referred to as Npc1 mice) were obtained from the Jackson Laboratory and maintained at the University of New Mexico Health Sciences Center in accordance with the Institutional Animal Care and Use Committee (Animal Welfare Assurance A3350-01 and U.S. Department of Agriculture Registration 85-R-0014). These mice were bred to generate Npc1 homozygous normal mice (Npc1+/+) and Npc1 heterozygous mice (Npc1+/−). The Npc1+/− mice have been previously characterized with decreased gene dosage and express one-half the normal amounts of functional NPC1 protein in all tissues and isolated fibroblasts (10, 42). The Npc1+/+ and Npc1+/− mice were housed in a room maintained at 24–25°C, 31–32% humidity, and alternating 12:12-h light-dark cycle.
Mouse diets.
At the time of weaning (4 wk) the male Npc1+/+ and Npc1+/− mice were fed either a low-fat diet (4.0% g fat; D07021301) or a high-fat diet (24% g fat; D07021302) with water ad libitum. This low-fat diet (LFD) and HFD are energy-balanced diets (same total % energy) formulated and produced by Research Diets. A semisynthetic HFD containing 5% fat in the form of sucrose polybehenate (D07031502) was also formulated to measure intestinal fat absorption. The composition and fatty acid profile of these mouse diets will be provided upon request.
Growth data and body weight analyses.
The growth data analysis represents repeated measures on body weights of 58 male mice (29 Npc1+/+ mice and 29 Npc1+/− mice) from 4 to 20 wk, as previously described (23). The mice were selected from 27 different litters (varying in size from 2 to 10 mice) on the basis of an Npc1+/+ or Npc1+/− genotype and randomized among 18 cages to receive a LFD or HFD. The fitted growth trajectories for the four groups of mice were modeled using a mixed-effects regression model fitted to the weight data over time using Stata Statistical Software, as previously described (44). The specific model used for the fitted growth trajectories is provided online in Supplemental Table S1. The body weight analyses represent different measures on the same groups of mice for body weights at 20 wk, change of body weights from 4 to 20 wk, and terminal body weights at 30 wk.
Physical and biochemical analyses.
The mice were food deprived (4–5 h) and euthanized using CO2 asphyxiation at 30 wk to obtain terminal body weights, tissue weights, and plasma. The collected tissues and plasma were frozen in liquid nitrogen for molecular and biochemical analyses. The liver lipids were extracted using an organic solvent, separated using thin-layer chromatography, and the amount of lipids (cholesterol, cholesteryl ester, free fatty acid, and triacylglycerol) was measured, as previously described (20). The amounts of liver lipids were normalized to the amount of liver protein precipitated during extraction to determine tissue concentration. The concentration of plasma cholesterol, triacylglycerol, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were determined using the Infinity Cholesterol, Triglyceride, ALT, and AST kits (Thermo Scientific), as previously described (20). The concentration of energy-regulating hormones (insulin and cortisol) and cytokines (IL-1β, IL-6, and TNF-α) were determined using the appropriate mouse insulin ELISA kit (ALPCO), cortisol EIA kit (Cayman Chemical), and Ready-SET-Go cytokine kits (eBioscience). The normal control serum (Data-Trol N) and abnormal control serum (Data-Trol A) (Thermo Scientific) were used as negative and positive controls, respectively.
Metabolic phenotype analyses.
A separate group of Npc1+/+ and Npc1+/− mice were generated and sent to the Seattle Mouse Metabolic Phenotyping Center at the University of Washington Health Sciences Center to perform comprehensive metabolic phenotype analyses. The intake of food and water, and determinants of energy metabolism (oxygen consumption, carbon dioxide production, locomotor activity, and heat production) during the 12:12-h light-dark cycles were measured for mice fed a LFD at 10 wk and for the same mice after they were fed a HFD for 9 days. These analyses were performed at an intermediate age (10–11 wk instead of 30 wk) to prevent confounding variables resulting from excessive weight gain and onset of metabolic disease. The rates of oxygen consumption, carbon dioxide production, locomotor activity, and heat production were measured continuously by indirect calorimetry over 36 h using the Comprehensive Laboratory Animal Monitoring System (Columbus Instruments).
Intestinal lipid absorption and fecal lipid content.
The noninvasive measurement of intestinal lipid absorption was measured as previously described (19). A separate group of Npc1+/+ and Npc1+/− mice were fed a LFD until 10 wk and a HFD containing 5% fat in the form of sucrose polybehenate (a nonabsorbable food additive) for 4 days with collection of fecal pellets on the past 2 days for analysis. Lipid absorption was calculated from the ratio of behenic acid to other fatty acids present in the diet, and feces were analyzed using gas chromatography of fatty acid methyl esters at the Cincinnati Mouse Metabolic Phenotypic Center at the University of Cincinnati. The same group of mice was then fed a HFD for 10 days with collection of fecal pellets for gravimetric analysis, as previously described (43). The fecal lipids were extracted using hexane:isopropanol (3:1) and allowed to dry. The dried lipid extract was suspended into 1 ml of chloroform containing 2% Triton X-100 and evaporated overnight. The pellet residue was suspended into 0.5 ml of water and solubilized overnight at 4°C. The lipid content (cholesterol, triacylglycerol, and free fatty acids) was measured using the Infinity Cholesterol and Infinity Triglyceride kits (Thermo Scientific), and the EnzyChrom free fatty acid kit (BioAssay Systems).
Measurement of adipose triacylglycerol lipolysis.
A separate group of Npc1+/+ and Npc1+/− mice were fed a HFD until 10 wk. Adipose triacylglycerol lipolysis was measured using inguinal white adipose tissue explants. The excised tissue was equilibrated overnight in DMEM and cut into portions (0.10–0.15 g) followed by incubation in DMEM with or without 1.0 μM isoproterenol for 3 h to activate the adipose tissue β-adrenergic receptor and triacylglycerol lipolysis (1). After incubation, the glycerol content of the medium was measured using the free glycerol reagent quantitative enzymatic assay (Sigma).
Cellular glycolysis metabolism and substrate oxidative metabolism.
Mouse fibroblasts were derived from the peritoneal membrane of Npc1+/+ and Npc1+/− mice at 4 wk and maintained in basic media (DMEM, 5.5 mM glucose, and 10% FBS). Fibroblasts were seeded into 24-well culture plates (5×105 cells/well) and incubated for 24 h before being placed into the SeaHorse XF24 extracellular analyzer used to determine the 1) extracellular acidification rate (ECAR), which is a measure of glycolytic metabolism (flux of glucose through the glycolysis pathway with production of pyruvate) and 2) oxygen consumption rate (OCR), which is a measure of substrate (glucose or fatty acid) oxidative metabolism (production of acetyl-CoA and flux through the citric acid cycle with production of reducing equivalents (NADH and FADH) that contribute to the electron transport chain for respiration. To determine OCR of endogenous fatty acids stored in the form of triacylglycerol, basic media were exchanged for substrate-limited media (DMEM, 0.5 mM glucose, 0.5 mM carnitine, and 1.0 FBS), and fibroblasts were incubated in the presence (control) or absence (treatment) of etomoxir (an inhibitor of carnitine palmitoyltransferase that facilitates the transfer of fatty acids from the cytoplasm across the inner mitochondrial membrane). In contrast, to determine OCR of exogenous fatty acids provided in media and conjugated to BSA, basic media were exchanged for substrate-limited media, and fibroblasts were incubated in the presence of BSA without fatty acids (control) or BSA with fatty acids (treatment) in the absence of etomoxir. The SeaHorse XF24 Extracellular Analyzer was run using an 8-min cyclic protocol command (mix for 3 min, stand 2 min, and measure for 3 min) (46, 47).
Western blot analysis.
The relative amounts of protein were determined using Western blot analysis, as previously described (20). The primary antibodies to detect mature sterol regulatory element-binding protein-1 (SREBP-1) and sterol regulatory element-binding protein-2 (SREBP-2) protein were purchased from Cayman Chemical (10007663) and Novus Biologicals (100–2215), respectively. The primary antibody to detect NPC1 protein was custom produced and affinity purified (Invitrogen). The primary antibodies to detect phosphorylated hormone-sensitive lipase (pHSL) and hormone-sensitive lipase (HSL) were purchased from Cell Signaling Technology (4139 and 4107). The antibodies to detect GAPDH and β-actin used as loading controls were purchased from Abcam (ab8245) and Sigma (A5316), respectively.
Statistical analyses.
Two-way ANOVA was conducted to examine the effect of Npc1 genotype and diet for the following measures: body weights at 20 wk, change in body weights from 4 to 20 wk, terminal body weights at 30 wk, tissue (liver and adipose) weights, and amounts of liver NPC1 protein, SREBP-1 protein, and SREBP-2 protein. Partial eta-squared (η2) statistics were used to measure effect size of the components in the model (genotype, diet, and interaction) and, thus, evaluate relative impact with a specific measure. One-way ANOVA was conducted for post hoc tests of mean differences in the metabolic phenotype measures for Npc1+/+ and Npc1+/− mice fed a LFD at 10 wk and after being fed a HFD for 9 days. Statistical analyses were performed using IBM SPSS version 24 with significance set at P < 0.05. Quantitative data (except growth data analysis) are represented as the means ± SE within a group of mice, tissues, or fibroblasts.
RESULTS
Growth data analysis and glucose tolerance test.
The Npc1+/+ and Npc1+/− mice fed a LFD or HFD were used for growth data analysis and glucose tolerance tests to quantitatively define the Npc1 gene-diet interaction in relation to weight gain and glucose tolerance. Fitted growth trajectories were derived from our original growth data for Npc1+/+ and Npc1+/− mice fed a LFD or HFD from 4 to 20 wk. The Npc1+/+ and Npc1+/− mice fed a LFD had no significant difference in growth trajectories (Fig. 1A). In contrast, the Npc1+/− mice fed a HFD had a significantly increased growth trajectory compared with Npc1+/+ mice fed the same diet. With respect to the glucose tolerance test performed at 20 wk, the Npc1+/+ and Npc1+/− mice fed a LFD had no significant difference in glucose tolerance (Fig. 1B). However, the Npc1+/+ and Npc1+/− mice fed a HFD had significantly impaired fasting glucose and glucose tolerance compared with Npc1+/+ and Npc1+/− mice fed a LFD. There was no significant difference in fasting glucose and glucose tolerance for Npc1+/+ and Npc1+/− mice fed a HFD. Therefore, the Npc1+/− mice fed a HFD had a significantly increased growth trajectory, but similar impaired fasting glucose and glucose tolerance compared with Npc1+/+ mice fed the same diet, confirming the Npc1 gene-diet interaction in relation to weight gain.
Fig. 1.
Growth data analysis and glucose tolerance test. A: growth data analysis using fitted growth trajectories for the Npc1+/+ and Npc1+/− mice fed a low-fat diet (LFD) or high-fat diet (HFD) from 4 to 20 wk. The model estimation accounts for sources of variation. B: glucose tolerance test for Npc1+/+ and Npc1+/− mice fed a LFD or HFD at 20 wk. Values are expressed as means ± SE of 14–15 mice per group. *P < 0.05 compared with Npc1+/+ fed a LFD.
Body weight analyses.
The Npc1+/+ and Npc1+/− mice fed a LFD or HFD were used for body weight analysis at 20 wk and also change of body weight analysis from 4 to 20 wk to further quantitatively define the Npc1 gene-diet interaction in relation to weight gain. A two-way ANOVA indicated a significant Npc1 gene-diet interaction for body weights at 20 wk. This analysis revealed significant main effects of Npc1 genotype and diet combined with the interaction accounted for an estimated 48% of variance in body weights. The partial eta-squared (η2) statistics estimated the relative impact of Npc1 genotype as 0.09, diet as 0.41, and interaction as 0.18. A two-way ANOVA also indicated a significant Npc1 gene-diet interaction for change of body weights from 4 to 20 wk. Although this analysis revealed no significant main effect of Npc1 genotype, there was a significant main effect of diet that combined with the interaction accounted for an estimated 40% of variance in change of body weights. The partial η2 statistics estimated the relative impact of diet as 0.33 and interaction as 0.21. Consistent with the growth data analysis, the Npc1+/+ and Npc1+/− mice fed a LFD had no significant difference in body weights at 20 wk (Fig. 2A). In contrast, the Npc1+/− mice fed a HFD had significantly increased body weights (13%) and also significantly increased change in body weights (18%) compared with Npc1+/+ mice fed the same diet (Fig. 2B). Therefore, the Npc1+/− mice fed a HFD had significantly increased body weights at 20 wk and change of body weights from 4 to 20 wk compared with Npc1+/+ mice fed the same diet, again confirming the Npc1 gene-diet interaction in relation to weight gain.
Fig. 2.
Body weight analyses. A: body weight analysis for Npc1+/+ and Npc1+/− mice fed a LFD or HFD at 20 wk. B: change of body weight analysis for Npc1+/+ and Npc1+/− mice fed a LFD or HFD at 4 and 20 wk. Values are expressed as means ± SE of 14 or 15 mice per group. *P < 0.05 compared with Npc1+/+ fed a HFD. **P < 0.05 compared with Npc1+/+ fed a LFD.
Amounts of liver NPC1 protein.
A schematic representation is provided for the normal and nonfunctional truncated NPC1 protein (Fig. 3A). The NPC1 protein antibody recognition site located near the carboxyl terminus is absent from the truncated NPC1 protein and, therefore, not detected using the NPC1 antibody. A two-way ANOVA indicated a significant Npc1 gene-diet interaction for amounts of liver NPC1 protein. This analysis revealed significant main effects of Npc1 genotype and diet that combined with the interaction accounted for an estimated 63% of variance in amounts of liver NPC1 protein. The partial η2 statistics estimated the relative impact of Npc1 genotype as 0.61, diet as 0.23, and interaction as 0.18. The amounts of NPC1 protein from Npc1+/− mice fed a HFD were significantly decreased compared with Npc1+/− mice fed a HFD (Fig. 3, B and C).
Fig. 3.
Amounts of liver NPC1 protein. A: schematic representation of the normal and truncated NPC1 protein. A retroposon insertion terminates protein translation producing a nonfunctional truncated NPC1 protein responsible for the null mutation. The transmembrane domains are indicated by vertical bars and the NPC1 protein antibody recognition site is indicated by an arrow near the carboxyl terminus. B: representative Western blot analysis of liver NPC1 protein and β-actin for Npc1+/+ and Npc1+/− mice fed a LFD or HFD at 30 wk C: amounts of liver NPC1 protein adjusted for β-actin and normalized to amounts of liver NPC1 protein for Npc1+/+ mice fed a LFD. Values are expressed as means ± SE of six mice per group. *P < 0.05 compared with Npc1+/+ mice fed a HFD.
Amounts of liver SREBP-1 and SREBP-2 protein.
A two-way ANOVA indicated no significant Npc1 gene-diet interaction for amounts of liver SREBP-1 protein. However, this analysis revealed significant main effects of Npc1 genotype and diet that combined accounted for an estimated 28% of variance in amounts of liver SREBP-1 protein. The partial η2 statistics estimated the relative impact of Npc1 genotype as 0.21 and diet as 0.22. The amounts of SREBP-1 protein in livers from Npc1+/− mice fed a LFD was significantly increased (118%) compared with Npc1+/+ mice fed a LFD (Fig. 4, A and B). The amounts of SREBP-1 protein in livers from Npc1+/− mice fed a HFD were not significantly different compared with Npc1+/+ mice fed a HFD. A two-way ANOVA indicated no significant Npc1 gene-diet interaction for amounts of liver SREBP-2 protein and no significant main effects of Npc1 genotype and diet (Fig. 4, C and D).
Fig. 4.
Amounts of liver SREBP-1 and SREBP-2 protein. A: representative Western blot analysis of liver SREBP-1 protein and β-actin for Npc1+/+ and Npc1+/− mice fed a LFD or HFD at 30 wk. B: amounts of liver SREBP-1 protein adjusted for β-actin and normalized to amounts of liver SREBP-1 protein for Npc1+/+ mice fed a LFD. C: representative Western blot analysis of liver SREBP-2 protein and β-actin for Npc1+/+ and Npc1+/− mice fed a LFD or HFD at 20 wk. D: amounts of liver SREBP-2 protein adjusted for β-actin and normalized to the amounts of liver SREBP-2 protein for Npc1+/+ mice fed a LFD. Values are expressed as means ± SE of six mice per group. *P < 0.05 compared with Npc1+/+ mice fed a LFD.
Physical and biochemical analyses.
A two-way ANOVA indicated a significant Npc1 gene-diet interaction for body weights at 30 wk. Although this analysis revealed no significant main effect of Npc1 genotype, there was a significant main effect of diet that combined with the interaction accounted for an estimated 54% of variance in body weights. The partial η2 statistics estimated the relative impact of diet as 0.48 and interaction as 0.23. The Npc1+/− mice fed a HFD had significantly increased body weights (13%) compared with Npc1+/+ mice fed the same diet at 30 wk (Table 1). A two-way ANOVA indicated a significant Npc1 gene-diet interaction for liver weights at 30 wk. This analysis revealed significant main effects of Npc1 genotype and diet that combined with the interaction accounted for an estimated 60% of variance in liver weights. The partial η2 statistics estimated the relative impact of Npc1 genotype as 0.21, diet as 0.50, and interaction as 0.27. The Npc1+/− mice fed a HFD had significantly increased liver weights (27%) compared with Npc1+/+ mice fed the same diet. A two-way ANOVA also indicated a significant Npc1 gene-diet interaction for adipose weights at 30 wk. Although this analysis revealed no significant main effect of Npc1 genotype, there was a significant main effect of diet that combined with the interaction accounted for an estimated 79% of variance in adipose weights. The partial η2 statistics estimated the relative impact of diet as 0.79 and interaction as 0.11. The Npc1+/− mice fed a HFD also had significantly increased adipose weights (14%) compared with Npc1+/+ mice fed the same diet. The Npc1+/− mice fed a HFD had significantly increased concentrations of liver cholesterol (24%) and liver triacylglycerol (39%) compared with Npc1+/+ mice fed the same diet. In support of these results, Npc1+/− mice fed a HFD had increased expression of genes encoding proteins for the integrated PGC-1α/β, LXRα/β, and SREBP-1 pathway (but not SREBP-2 pathway), glycolysis pathway, and lipogenesis pathway compared with Npc1+/+ mice fed the same diet, as determined using RNA microarray analysis (Table 2). With respect to plasma components, Npc1+/− mice fed a HFD had significantly increased concentrations of cortisol (10%), insulin (82%), and ALT (59%) compared with Npc1+/+ mice fed the same diet. The increased concentrations of cortisol and insulin are known to affect regulation of the SREBP-1, glycolysis, and lipogenesis pathways to promote positive energy balance. Therefore, Npc1+/− mice fed a HFD had significantly increased body weights, tissue weights (liver and adipose), and plasma components compared with Npc1+/+ mice fed the same diet, again confirming the Npc1 gene-diet interaction in relation to weight gain.
Table 1.
Physical and biochemical measures for Npc1+/+ and Npc1+/− mice fed a LFD or HFD
| +/+ LFD | +/− LFD | +/+ HFD | +/− HFD | |
|---|---|---|---|---|
| Physical measures | ||||
| Body weights, g | 32.5 ± 0.5 | 30.8 ± 0.8 | 34.8 ± 0.8 | 39.2 ± 0.9* |
| Liver weights, g | 1.48 ± 0.04 | 1.44 ± 0.04 | 1.64 ± 0.05 | 2.08 ± 0.07* |
| Adipose weights, g | 0.57 ± 0.05 | 0.49 ± 0.04 | 1.11 ± 0.06 | 1.26 ± 0.04* |
| Liver concentrations | ||||
| C, nmol/mg protein | 7.64 ± 0.52 | 7.08 ± 0.25 | 7.25 ± 0.34 | 8.96 ± 0.18* |
| CE, nmol/mg protein | 4.88 ± 0.44 | 5.10 ± 1.07 | 8.94 ± 1.46 | 8.56 ± 0.55 |
| FFA, nmol/mg protein | 5.43 ± 1.71 | 7.29 ± 1.40 | 14.7 ± 0.7 | 15.9 ± 1.2 |
| TAG, nmol/mg protein | 260 ± 10 | 363 ± 70* | 618 ± 36 | 858 ± 38* |
| Plasma concentrations | ||||
| TAG, mmol/l | 1.51 ± 0.12 | 1.19 ± 0.16 | 1.18 ± 0.07 | 1.06 ± 0.09 |
| TC, mmol/l | 4.04 ± 0.30 | 4.13 ± 0.22 | 4.56 ± 0.16 | 4.12 ± 0.22 |
| Cortisol, ng/l | 4.47 ± 0.19 | 4.50 ± 0.19 | 5.2 ± 0.18 | 5.72 ± 0.18* |
| Insulin, mg/l | 1.43 ± 0.13 | 1.09 ± 0.10 | 1.48 ± 0.18 | 2.70 ± 0.30* |
| IL-1β, ng/l | 26.4 ± 2.1 | 25.8 ± 1.5 | 26.2 ± 1.7 | 23.3 ± 2.5 |
| IL-6, ng/l | 34.1 ± 8.4 | 35.5 ± 8.9 | 36.9 ± 4.6 | 36.7 ± 2.6 |
| TNF-α, ng/l | 28.5 ± 5.1 | 47.4 ± 8.9 | 21.4 ± 2.0 | 23.2 ± 4.0 |
| AST, U/l | 152 ± 32 | 168 ± 12 | 138 ± 25 | 171 ± 22 |
| ALT, U/l | 79 ± 23 | 97 ± 17 | 162 ± 30 | 258 ± 22* |
Values are expressed as means ± SE for 14 or 15 mice per group at 30 wk. ALT, alanine aminotransferase; AST, aspartate aminotransferase; C, cholesterol; CE, cholesterol ester; FFA, free fatty acid; TC, total cholesterol; TAG, triacylglycerol. LFD, low-fat diet; HFD, high-fat diet; Npc1, Niemann-Pick C1 gene.
P < 0.05 compared with Npc1+/+ mice fed a HFD.
Table 2.
RNA microarray of livers from Npc1+/+ and Npc1+/− mice fed a HFD
| Gene Product | Abbreviation | Fold Δ |
|---|---|---|
| Sterol regulatory element-binding protein pathway | ||
| Peroxisome proliferator-activated receptor γ coactivator 1-α | Pgc1a | 1.38* |
| Peroxisome proliferator-activated receptor γ coactivator 1-β | Pgc1b | 0.93 |
| Liver X receptor α | Lxra | 1.25* |
| Liver X receptor β | Lxrb | 1.18 |
| Retinoid X receptor α | Rxra | 1.28 |
| Insulin-induced gene 1 | Insig1 | 1.13 |
| Insulin-induced gene 2 | Insig2 | 0.75 |
| Sterol regulatory element-binding protein cleavage activating protein | Scap | 1.13 |
| Sterol regulatory element-binding protein-1 | Srebp1 | 1.98* |
| Sterol regulatory element-binding protein-2 | Srebp2 | 1.00 |
| Peroxisome proliferator-activated receptor pathway | ||
| Peroxisome proliferator-activated receptor α | Ppara | 0.58* |
| Peroxisome proliferator-activated receptor δ | Ppard | 1.00 |
| Peroxisome proliferator-activated receptor γ | Pparg | 0.89 |
| Glycolysis pathway | ||
| Glucose kinase | Gck | 2.21* |
| Glucose kinase regulatory protein | Gckr | 1.30* |
| Phosphoglucomutase | Pgm | 0.93 |
| Phosphofructokinase | Pfk | 1.23 |
| Aldolase | Aldo | 1.10 |
| Triose phosphate mutase | Tpm | 1.04 |
| Glyceraldehyde 3-phosphate dehydrogenase | Gapdh | 1.06 |
| Phosphoglycerate kinase | Pgk | 1.00 |
| Phosphoglycerate isomerase | Pgam | 1.00 |
| Enolase | Enoa | 1.00 |
| Pyruvate kinase | Pklr | 1.39* |
| Tricarboxylic acid cycle pathway | ||
| Pyruvate dehydrogenase | Pdh | 1.46* |
| Citrate synthase | Cs | 1.25* |
| Aconitase | Aco | 1.14 |
| Isocitrate dehydrogenase | Idh | 0.98 |
| Ketoglutarate dehydrogenase | kgdh | 1.00 |
| Succinyl-CoA synthetase | Scs | 1.00 |
| Succinate dehydrogenase | Sdha | 1.05 |
| Fumarate dehydratase | Fd | 1.06 |
| Malate dehydrogenase | Mdh | 1.00 |
| Lipogenesis pathway | ||
| ATP citrate lyase | Acly | 0.96 |
| Acetyl-CoA carboxylase | Acac | 1.28* |
| Fatty acid synthase | Fasn | 1.80* |
| Steroyl-CoA desaturase | Scd | 1.51* |
| 3-hydroxy-3-methylglutaryl-CoA synthase | Hmgcs | 1.00 |
| 3-hydroxy-3-methylglutaryl-CoA reductase | Hmgcr | 1.59 |
| Malic enzyme | Mod | 1.16 |
| Glucose 6-phosphate dehydrogenase | G6pdh | 1.00 |
| Phosphogluconate dehydrogenase | Pgd | 1.25* |
Values represent the fold change (increased or decreased) of mRNA levels for Npc1+/− mice compared with Npc1+/+ fed a HFD.
An asterisk indicates at least a 1.25-fold increase or decrease in amounts of mRNA (mean value of three mice per group) and represents those genes previously reported to be upregulated or downregulated by the SREBP-1 pathway (29).
Metabolic phenotype analyses.
Comprehensive metabolic phenotype analyses was performed using a separate group of Npc1+/+ and Npc1+/− mice fed a LFD at 10 wk and for the same mice after they were fed a HFD for 9 days (thus, measures were obtained at 10 wk and ~11 wk). The results indicated no significant differences for any measures (intake of food or water, oxygen consumption, carbon dioxide elimination, locomotor activity, and heat production) when comparing Npc1+/+ and Npc1+/− mice fed a LFD or HFD during both the light and dark cycles (data not shown).
Intestinal lipid absorption and fecal lipid content.
Intestinal lipid absorption and fecal lipid content were measured to determine whether increased absorption might account for weight gain. The results indicated no significant difference of intestinal lipid absorption for Npc1+/+ and Npc1+/− mice fed a LFD at 10 wk and the same mice fed a HFD-sucrose polybehenate for 3 days (data not shown). Moreover, these same mice fed a HFD for 10 days had no significant difference in the concentration of total fecal lipid content or fecal lipid composition (cholesterol, triacylglycerol, and fatty acids) (data not shown). Therefore, the Npc1+/− mice fed a HFD had no significant difference in lipid absorption or fecal lipid content compared with Npc1+/+ mice fed the same diet.
Adipose lipolysis and amounts of phosphorylated hormone-sensitive lipase.
The amounts of glycerol released (a measure of triacylglycerol lipolysis and mobilization) from adipose of Npc1+/− mice fed a HFD at 10 wk was significantly decreased (19.9%) compared with adipose from Npc1+/+ mice fed the same diet (Fig. 5A). Moreover, the amounts of pHSL and pHSL/HSL (relative and adjusted amounts of activated HSL) from adipose of Npc1+/− mice fed a HFD were significantly decreased (52.1% and 50.4%, respectively) compared with adipose from Npc1+/+ mice fed the same diet (Fig. 5, B and C). Therefore, the Npc1+/− mice fed a HFD had decreased activation of HSL, decreased triacylglycerol lipolysis, and decreased glycerol mobilization compared with Npc1+/+ mice fed the same diet.
Fig. 5.
Adipose lipolysis and the amounts of phosphorylated hormone-sensitive lipase. A: release of glycerol (concentration of glycerol in the media) from adipose lipolysis for Npc1+/+ and Npc1+/− mice fed a HFD at 10 wk. B: representative Western blot analysis of adipose pHSL, HSL, and GAPDH protein for Npc1+/+ and Npc1+/− mice fed a HFD at 10 wk. C: amounts of adipose pHSL, HSL, and pHSL/HSL protein adjusted for GAPDH and normalized to the amounts of adipose pHSL, HSL, and pHSL/HSL protein for Npc1+/+ fed a HFD at 10 wk. Values are means ± SE of 8–10 mice per group for release of glycerol and five mice per group for Western blot analysis. *P < 0.05 compared with Npc1+/+ mice fed the same diet. HSL, hormone sensitive lipase; pHSL, phosphorylated HSL; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Cellular glycolysis and substrate oxidative metabolism.
The Npc1+/+ and Npc1+/− fibroblasts grown and assayed in basic media or glucose-limited media with lipid substrate were used to examine energy metabolism pathways (glycolysis, citric acid cycle, and lipolysis). The Npc1+/− fibroblasts grown and assayed in basic media had significantly increased glycolytic metabolism (31%) compared with Npc1+/+ fibroblasts grown and assayed in the same media (Fig. 6A). However, the Npc1+/− fibroblasts grown and assayed in basic media also had significantly decreased oxidative metabolism (46%) compared with Npc1+/+ fibroblasts grown and assayed in the same media (Fig. 6B). Moreover, the Npc1+/− fibroblasts grown and assayed in glucose-limited media (allowing endogenous fatty acids to serve as an energy substrate) had significantly decreased oxidative metabolism (93%) compared with Npc1+/+ fibroblasts grown and assayed in the same media (Fig. 6C). Finally, the Npc1+/− fibroblasts grown and assayed in glucose-limited media with lipid substrate (BSA conjugated fatty acids) had significantly increased oxidative metabolism (1174%) compared with Npc1+/+ fibroblasts grown and assayed in the same media (Fig. 6D). Therefore, the Npc1+/− fibroblasts have an increased capacity for glycolysis (glucose oxidation to produce pyruvate) but decreased capacity for converting pyruvate to acetyl-CoA for oxidation in the citric acid cycle and respiration using the electron transport chain compared with Npc1+/+ fibroblasts. However, the Npc1+/− fibroblasts had a decreased capacity for converting endogenous fatty acids but increased capacity for converting exogenous fatty acids to acetyl-CoA for oxidation in the citric acid cycle and respiration using the electron transport chain compared with Npc1+/+ fibroblasts.
Fig. 6.
Cellular glycolysis and substrate oxidative metabolism. A: basal glycolysis for Npc1+/+ and Npc1+/− fibroblasts grown and assayed in basic media. B: basal glucose oxidative metabolism for Npc1+/+ and Npc1+/− fibroblasts grown and assayed in basic media. C: basal endogenous fatty acid oxidative metabolism for Npc1+/+ and Npc1+/− fibroblasts grown in basic media and assayed in glucose-limited media. D: basal exogenous fatty acid oxidative metabolism for Npc1+/+ and Npc1+/− fibroblasts grown in basic media and assayed in glucose-limited media. Values are expressed as means ± SE of 15 wells for Npc1+/+ and Npc1+/− fibroblasts. *P < 0.05 compared with Npc1+/+ fibroblasts grown and assayed in the same media. ECAR, extracellular acidification rate; OCR, oxygen consumption rate.
Cellular growth curves, mitochondrial content, and metabolic adaptations.
The Npc1+/+ and Npc1+/− fibroblasts grown in basic media were used to examine cellular growth rates, mitochondrial content, and metabolic adaptations. The Npc1+/− fibroblasts grown in basic media had a significantly decreased growth rate (27%), as measured using cell confluence during 96 h compared with Npc1+/+ fibroblasts grown in the same media (Fig. 7A). Moreover, the Npc1+/− fibroblasts grown in basic media had a significantly decreased mitochondrial content (32%) and decreased concentration of ATP (53%) at 96 h compared with Npc1+/+ fibroblasts grown in the same media (Fig. 7, B and C). Finally, the Npc1+/− fibroblasts grown in basic media had significantly increased amounts of triacylglycerol (44%) and cholesterol (31%) at 96 h compared with Npc1+/+ fibroblasts grown in the same media (Fig. 7, D–F). Therefore, the Npc1+/− fibroblasts have a decreased growth rate and increased capacity for storage of neutral lipids (triacylglycerol and cholesterol) compared with Npc1+/+ fibroblasts.
Fig. 7.
Cellular growth curves, mitochondrial content, and metabolic adaptations. A: cellular growth curves for Npc1+/+ and Npc1+/− fibroblasts grown in basic media during 96 h. B: mitochondrial content for Npc1+/+ and Npc1+/− fibroblasts grown in basic media at 96 h. C: concentration of ATP for Npc1+/+ and Npc1+/− fibroblasts grown in basic media at 96 h. D: representative images of triacylglycerol staining for Npc1+/+ and Npc1+/− fibroblasts grown in basic media at 96 h. E: amounts of triacylglycerol for Npc1+/+ and Npc1+/− fibroblasts grown in basic media at 96 h. F: amounts of cholesterol for Npc1+/+ and Npc1+/− fibroblasts grown in basic media at 96 h. Values are expressed as means ± SE of 15 wells for Npc1+/+ and Npc1+/− fibroblasts. *P < 0.05 compared with Npc1+/+ fibroblasts grown in the same media.
DISCUSSION
The objective for our current study was to validate the Npc1 gene-diet interaction using statistical modeling with fitted growth trajectories, conduct body weight analyses for different measures, and define the physiological basis responsible for weight gain. First, the statistical modeling with fitted growth trajectories, accounting for sources of variation and two-way ANOVA of body weights at 20 wk and change of body weight from 4 to 20 wk, demonstrated that the Npc1 gene interacts with a HFD to promote weight gain. Second, although metabolic phenotype analysis indicated no significant difference between Npc1+/+ and Npc1+/− mice fed a HFD for food and water intake, oxygen consumption, carbon dioxide production, locomotor activity, adaptive thermogenesis, and intestinal lipid absorption, the livers from Npc1+/− mice had significantly increased amounts of mature SREBP-1 and increased expression of SREBP-1 target genes that regulated glycolysis and lipogenesis with an accumulation of triacylglycerol and cholesterol. Moreover, white adipose tissue from Npc1+/− mice had significantly decreased amounts of pHSL with decreased triacylglycerol lipolysis. Consistent with these results, cellular energy metabolism studies indicated that Npc1+/− fibroblasts had significantly increased glycolysis and lipogenesis, in addition to significantly decreased substrate (glucose and endogenous fatty acid) oxidative metabolism with an accumulation of triacylglycerol and cholesterol. Together, these studies demonstrated that the Npc1 gene interacts with a HFD to promote weight gain through differential regulation of central energy metabolism pathways.
A key finding of our studies is that Npc1+/− mice fed a LFD or HFD have an accumulation of liver triacylglycerol (hepatic steatosis) compared with Npc1+/+ mice fed the same diet at 30 wk, consistent with one of our previous studies showing that Npc1+/− mice fed a LFD develop hepatic steatosis at 5 wk in the absence of weight gain (12). These studies also indicated that Npc1+/− mice have impaired feedback inhibition (activation) of the liver SREBP-1 pathway, whereby increased amounts of SREBP-1 protein serves as a potent transcription factor for increasing hepatic glycolysis and lipogenesis (14). The physical phenotype resulting from impaired feedback inhibition of the SREBP-1 pathway has previously been characterized using mice that overexpress the Srebp-1 gene and human SREBP-1 gene gain-of-function variants reported to be associated with hepatic steatosis and weight gain (2, 8). Therefore, our results indicating that Npc1+/− mice fed a HFD have impaired feedback inhibition of the SREBP-1 pathway and increased expression of target genes encoding for proteins participating in glycolysis and lipogenesis represents the “liver component” for the Npc1 gene-diet interaction responsible for weight gain. Moreover, our results indicating that Npc1+/− mice fed a HFD have decreased phosphorylation (decreased activation) of adipose HSL, which catalyzes hydrolysis of adipose triacylglycerol and mobilization of fatty acids, represent the “adipose component” of the Npc1 gene-diet interaction responsible for weight gain. These tissue-specific alterations in regulation of energy metabolism pathways in combination with Npc1+/− mice fed a HFD having increased expression of the PGC-1α/β transcription coactivators are consistent with other studies demonstrating that a HFD increases expression of the PGC-1α/β transcriptional coactivators to potentiate LXR nuclear receptor transcriptional activity and Srebp-1 gene expression to differentially regulate tissue lipogenesis and lipolysis (16, 27, 34, 35). Indeed, the Npc1+/− mice that were fed a HFD had significantly increased concentrations of plasma cortisol and insulin, known to increase both liver and adipose lipogenesis, while at the same time inhibiting adipose lipolysis in the presence of insulin resistance (7, 38).
The increased liver lipogenesis and decreased adipose lipolysis was phenotypically similar to our additional studies performed using Npc1+/+ and Npc1+/− fibroblasts, indicating altered cellular energy metabolism of glucose and fatty acids. These studies indicated that Npc1+/− fibroblasts have increased glycolysis but decreased substrate (glucose and endogenous fatty acid) oxidative metabolism compared with Npc1+/+ fibroblasts grown in the same media. Moreover, the Npc1+/− fibroblasts also have decreased oxidative metabolism of endogenous fatty acids but increased oxidative metabolism of exogenous fatty acids compared with Npc1+/+ fibroblasts grown in the same media. The decreased substrate oxidative metabolism of glucose may be attributed to the decreased mitochondrial content, thereby diverting flux of acetyl-CoA into the synthesis of neutral lipids. This particular result is consistent with another study indicating that NPC1 deficiency leads to alterations in mitochondrial function and energy metabolism, including increased glycolysis and decreased oxidative metabolism (25). The decreased oxidative metabolism of endogenous fatty acid in the form of triacylglycerol suggests a deficiency in triacylglycerol lipolysis, particularly since oxidative metabolism of exogenous fatty acid is capable of compensating for this deficiency, as evidenced by increased oxidative metabolism. A schematic representation of the proposed physiological basis for the Npc1 gene-diet interaction that promotes weight gain is provided (Fig. 8).
Fig. 8.
Schematic representation of the proposed Npc1 gene-diet metabolic pathway that promotes weight gain. A: dietary saturated fatty acids have been reported to increase expression of the Pgc1a and Pgc1b genes and the amounts of encoded PGC-1α and PGC1-β proteins. B: the PGC-1α and PGC1-β proteins serve as transcriptional coactivators for the nuclear receptor LXR (activated in the presence of oxysterol) that forms a heterodimer with RXR (activated in the presence of retinoic acid) to increase expression of the Srebp1 gene and the amounts of encoded precursor SREBP-1 protein. C: decreased Npc1 gene dosage and decreased amounts of encoded NPC1 protein has been reported to impair feedback inhibition of the SREBP pathway characterized by an increased amount of mature SREBP-1 protein that serves as a transcription factor. D: the increased amounts of PGC-1β protein (but not PGC-1α protein) have also been reported to serve as a transcriptional coactivator for the increased amounts of mature SREBP-1 protein to increase expression of target genes encoding proteins that regulate increased glycolysis and lipogenesis but also decreased expression of the Ppara gene and the amounts of encoded PPARα protein. E: the PGC-1α protein (but not PGC-1β protein) serves as a transcriptional coactivator for the decreased amounts of PPARα protein that forms a heterodimer with RXR and decreases expression of target genes. F: these PPARα target genes encode proteins that regulate triacylglycerol lipolysis and fatty acid β-oxidation. Therefore, the proposed Npc1 gene-diet interaction metabolic pathway promotes weight gain through increased glycolysis and lipogenesis in addition to decreased triacylglycerol lipolysis and fatty acid β-oxidation. PGC-1α, peroxisome proliferator-activated receptor gamma coactivator-1α; PGC-1β peroxisome-proliferator activated receptor gamma coactivator-1β; LXR, liver X receptor; RXR, retinoid X receptor; LXRE, liver X receptor response element; SREBP-1, sterol regulatory element-binding protein-1; SREBP, sterol regulatory element-binding protein response element; PPARα, peroxisome-proliferator activated receptor alpha; PPRE, peroxisome-proliferator activated receptor response element.
In summary, the present study has shown that Npc1+/− mice with decreased gene dosage interacts with a HFD to promote positive energy balance and weight gain compared with Npc1+/+ mice fed the same diet. The physiological basis for the Npc1 gene-diet interaction indicates that impaired feedback inhibition of the SREBP-1 pathway increases liver glycolysis and lipogenesis, in addition to decreases in adipose lipolysis, thereby resulting in an accumulation of neutral lipids in both tissues. These results were similar to studies performing with Npc1+/+ and Npc1+/− fibroblasts, indicating an increased flux of acetyl-CoA through the lipogenesis pathway to promote storage of neutral lipids in combination with deficient lipolysis and oxidative metabolism of endogenous fatty acids in the form of triacylglycerol. In conclusion, these studies demonstrate that the Npc1 gene interacts with a HFD to promote weight gain through differential regulation of central energy metabolism pathways.
GRANTS
This work was supported in part by National Institutes of Health (NIH) Grant DK-071544, NIH grant DK-076126 (MMPC at the University of Washington Health Sciences Center), NIH grant DK-059630 (MMPC at the University of Cincinnati Health Sciences Center), and private funding through the University of New Mexico Foundation for the investigation of genetic and metabolic diseases.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
J.J.C., D.J., H.W., N.P.G., R.A.V., R.G.-S., and W.S.G. performed experiments; J.J.C., D.J., H.W., N.P.G., L.J.H., F.J.M., R.G.-S., K.A.T., R.A.H., D.M., R.A.O., R.C.L., and W.S.G. analyzed data; J.J.C., D.J., H.W., N.P.G., L.J.H., F.J.M., K.A.T., R.A.H., D.M., R.A.O., R.C.L., and W.S.G. interpreted results of experiments; J.J.C., D.J., and W.S.G. prepared figures; J.J.C., D.J., and W.S.G. drafted manuscript; J.J.C., D.J., H.W., N.P.G., R.A.V., L.J.H., F.J.M., R.G.-S., K.A.T., R.A.H., D.M., R.A.O., R.C.L., and W.S.G. edited and revised manuscript; J.J.C., D.J., H.W., N.P.G., R.A.V., L.J.H., F.J.M., R.G.-S., K.A.T., R.A.H., D.M., R.A.O., R.C.L., and W.S.G. approved final version of manuscript.
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