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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Gene. Author manuscript; available in PMC Jan 10, 2013.
Published in final edited form as:
PMCID: PMC3258670
NIHMSID: NIHMS336217

Genetic variation in the mouse model of Niemann Pick C1 affects female, as well as male, adiposity, and hepatic bile transporters but has indeterminate effects on caveolae

Abstract

We have previously shown that male Npc1 heterozygous mice (Npc1+/), as compared to homozygous wild-type mice (Npc1+/+), both maintained on the —lean BALB/cJ genetic background, become obese on a high fat but not on a low fat diet. We have now extended this result for female heterozygous mice. When fed high-fat diet, the Npc1+/ white adipose weight is also increased in females, therefore following the same trend as males. Bile transporters which had previously been found to be altered in Npc1/ mice on a high fat diet, showed related, but small, changes in mRNA levels but large changes in protein expression. We have addressed the possible role of caveolae in these differences. It has long been known that caveolin 1 is increased in the liver (sex not specified) of Npc1+/ (compared to Npc1+/+ and Npc1/) mice and in heterozygous cultured skin fibroblasts of NPC1 carriers. We now find that caveolin 1 is increased in male, but not female liver and female, but not male adipose tissue. The caveolin 1 increase was not accompanied by changes in another caveolar protein, polymerase1 and transcript release factor (Ptrf). The numbers of caveolae in female adipose cells could not be correlated with levels of caveolae. Thus, we conclude that Npc1 affects female as well as male obesity and bile transporters but that effects on caveolin 1 are not discernible.

Keywords: obesity, white adipose tissue, caveolin 1, polymerase 1 and transcript release factor

1. Introduction

Niemann-Pick disease type C (NPC) is a neurodegenerative disorder that usually presents in the first decade of life [Patterson, et al, 2001]. The classic presentation of NPC is a child of either sex developing coordination problems, dysarthria, and hepatosplenomegaly during early school-age years. The neurological progression of the disorder is relentless and is characterized by increasing severity of ataxia, developmental dystonia, and dementia, until death supervenes, usually during the second decade of life. Identification of the major gene responsible for the disorder, NPC1/Npc1, revealed one coding for a multipass transmembrane protein containing a sterol-sensing domain that shows homology to the genes for Patched, HMG CoA reductase, and SCAP (SREBP cleavage activating protein) [Carstea, et al, 1997; Loftus, et al, 1997]. Analysis of NPC1 protein function suggests that it is involved in late endosomal lipid sorting/vesicular trafficking [Garver, et al, 2000; Higgins, et al, 1999; Neufeld, et al, 1999]. The fundamental role of NPC1 in intracellular lipid transport, and the severe visceral disease sometimes causing death before the onset of neurodegeneration, established the idea that lipid accumulation is the primary defect in NPC and leads to secondary neurological impairment.

We have previously reported that mice homozygous recessive for Npc1 develop severe liver disease, with altered levels of bile transporters, when fed a high fat, 1% cholesterol diet [Erickson, et al, 2005]. We did not study the effects of heterozygosity for Npc1 at that time. Our interest in heterozygous effects was stimulated by Meyre, et al’s finding [2009] that common variants in the human gene were associated with early onset and morbid obesity. It was not known whether these variants increased or decreased NPC1 function. We found that heterozygous Npc1+/− male mice, when fed a high fat diet, deposited more fat and were heavier than their wild-type siblings [Jelinek, et al, 2010].

Previously we had also shown that caveolin 1 was increased in NPC1 variants, both man and mouse, mostly in heterozygotes, but to a lesser degree in homozygotes. There are multiple lines of evidence implicating caveolin 1 and caveolae in the regulation of lipid metabolism. The mouse knockout (KO) for caveolin 1 has a lean phenotype [Razinie, et al, 2002] and caveolin-1 is upregulated in adipose tissue from obese individuals [Catalan, et al, 2008]. Mechanisms for this relationship have been postulated. The adipocyte cell surface insulin receptor is found in caveolae [Gustavsson, et al, 1999]. Not only is the insulin receptor located in caveolae, but insulin-induced translocation of glucose transporter proteins (GLUT4) from intracellular stores to the plasma membrance leads to their location in caveolae [Scherer, et al, 1994]. Caveolin 1 deficient mice become insulin resistant [Cohen, et al, 2003]. The finding that hexokinase localizes with glucose transporters in caveolae suggests the possibility that glucose can also be metabolized in caveolae [Rauch, et al, 2006]. Another major class of metabolites, well-documented to be taken up by cells through caveolae, are fatty acids. Caveolin 1 itself is a fatty acid binding protein [Trigatti, et al, 1999]. The fatty acid transport proteins 1, 4 and fatty acid translocase are other proteins that bind fatty acids in caveolae [Rauch, et al, 2006; Ortegren, et al, 2006 Ring, et al, 2006]. Thus, we have also explored the possibility that increases in caveolin 1 found in heterozygous female adipose tissue could be causal for the adiposity phenotype by increasing the number of caveolae.

2. Materials and Methods

2.1 Mice

BALB/cJ Npc1nih heterozygous (Npc1+/) mice were bred to generate Npc1 wild-type (Npc1+/+) mice and Npc1 heterozygous (Npc1+/). The mice were maintained at the University of Arizona Animal Care Facility, with 12 hr light, 12 hr dark cycles and water ad libitum.

2.2 Genotype analysis

At the time of weaning (21 days), the Npc1 mice were genetically identified using PCR at the Npc1 locus as previously described [Loftus, et al, 1997]. In brief, PCR amplification was used with 20–40 ng of DNA (prepared from tail-tips) with 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 1.5% agarose gel and visualized with a UV light.

2.3 Study protocol

One week after weaning (28 days of age), the female Npc1+/+ and Npc1+/ mice were weighed and placed on either a regular/low-fat diet (18% kcal [6.2% wt.] fat, NIH-301) or high-fat diet (45% kcal [24% wt.] fat , Diet-07021302) produced by Research Diets (New Brunswick, NJ). 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 [4Garver, et al, 2000; Garver, et al, 2007]. The Npc1+/+ and Npc1+/ mice were weighed every week from 5 to 25 weeks of age. At about 30 weeks of age, the Npc1+/+ and Npc1+/ mice fed either the regular or the high-fat diet were killed and the liver weights and the ovarian fat pad weights were determined. Livers were than snap frozen in liquid N2.

2.4 Bile transporter gene expression

Multidrug resistance protein (Mrp) 1, Mrp2, Mrp 3, Mrp5, Na+-taurocholic co-transporting polypeptide (Ntcp), Bile salt export pump (Bsep), and Organic anion transport protein 2 (Oatp2) were measured by the branched DNA signal amplification test as previously described [Erickson, et al 2005]. Briefly, total RNA was prepared from snap-frozen liver using RNAzol B reagent (Tel-Test, Friendswood, TX). The quality of the RNA samples was judged by gel electrophoresis, monitoring the integrity and ratio of 28S and 18S bands. Using QuantiGene Discover kit (Genospectra, Fremon, CA) reagents, 10 ug of total RNA was hybridized overnight at 37 C in 96-well plates with blocker, capture and label probes for each transporter— sequences are provided in Erickson, et al [2005] and references therein. After post-hybridization washes, luminescence was measured with the Quantiplex 320 branched DNA Luminometer (Bayer Diagnostics) interfaced with the Quantiplex Data Management Software version 5.02 (Bayer Diagnostics).

2.5 Western blotting

Levels of bile transporters Ntcp, OatP2, and Mrp2 were determined in liver, caveolin 1 was determined in liver and white adipose tissue, and Ptrf only in white adipose tissue, of the Npc1+/ and Npc1+/+ females and Npc1+/+ males at 30 weeks of age, by Western blots. Whole cell lysate preparations of mouse liver tissue were prepared from tissue homogenized in NP-40 buffer (20 mM Tris HCl, 137 mM NaCl, 10% glycerol, 1% nonidet P-40, and 2 mM EDTA with 1 Protease Inhibitor Cocktail Tablet (Roche, Indianapolis, IN) per 50mL) at 4°C. Homogenized tissue was then agitated at 4°C for 2 hours, centrifuged at 10,000 x g for 30 minutes, and the supernatant transferred to a clean collection tube. The relative amounts of protein were determined using immunoblot analysis. Protein samples were separated using 7% (transporters) or 14% (caveolin 1 and Ptrf) SDS-PAGE under reduced conditions and then transferred to a PVDF (transporters) or nitrocellulose (caveolin 1 and Ptrf) membrane. In brief, blocking buffer (10mM sodium phosphate pH 7.4, 150 mM NaCl, 0.05% Tween 20, and 5% non-fat dry milk) was used to block non-specific sites on the nitrocellulose membrane (2h). The membranes were then incubated in blocking buffer containing the appropriate dilution of primary antibody (4ºC for 16 hr). The membranes were rinsed with blocking buffer (3 x 10 min) to remove residual primary antibody and then incubated in blocking buffer containing the appropriate dilution of peroxidase-conjugated secondary antibody (90 min). The membranes were rinsed with blocking buffer (3 x 10 min) to remove residual secondary antibody and enhanced chemiluminescence (ECL) was performed to obtain autoradiograms. The relative amounts of caveolin 1 and Ptrf protein and endogenous control protein (β-actin) were quantified within the linear range of film using a BioRad Model GS-700 Imaging Densitometer. The antibody for caveolin 1 (clone pAb) was purchased from BD Biosciences (San Jose, CA) and the antibody for Ptrf was from Abcam (Cambridge, MA). For bile transporters, the following antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used to determine relative protein levels: Mrp2 (H-17, 1:1000), Ntcp (M-130, 1:5000), and Oatp2 (N-16, 1:1000). Quantification of relative protein expression was determined using image processing and analysis with Image J software (NIH, Bethesda, MD) and normalized to Gapdh (1:7000, Santa Cruz Biotechnology, Santa Cruz, CA).

2.6 Transmission electron microscopy

Portions of the adipose tissue samples from 30 week old females were minced to several mm2 cubes and fixed in Karnovsky’s. After overnight fixation, they were rinsed in Karnovsky’s storage buffer. The specimens were flat embedded in eponate resin. Areas of interest were cut and mounted on cylinders for further sectioning and imaging. Numbers of caveolae were determined by manual counts per areas ranging from 1,850 – 2,100 μM2. They were expressed per 670 μM2.

3. Results

3.1 Weight gain in female mice

No significant differences in weight curves were found between female Npc1+/ and Npc1+/+ mice when fed a regular diet (18% of calories from fat) although there was a trend for them to be lighter at 19–23 weeks of age (Fig 1A). However, when placed on the high fat diet (45% of calories from fat), female Npc1+/ were heavier than female Npc1+/+ mice from week 9 onwards and the difference had become significant from 18 weeks onwards (Fig 1B). At 30 weeks of age, the Npc1+/− mice on the high fat diet had a significantly increased (23.3%, p = 0.0022) body weight compared to Npc1+/+ mice fed the same high-fat diet. This was associated with increased white adipose tissue weight but not liver weight (Table 1). The females on the regular diet did not show differences in these parameters.

Fig 1
Growth curves for female Npc1+/+ and Npc1+/− mice from 5 to 25 weeks of age when fed a regular (18% kcal fat) diet (A) and a high-fat (45% kcal fat) diet (B). The groups were represented with 6-13 mice. * p ≤ 0.05 when compared to Npc1 ...

3.2 Bile transporter expression

Of the 7 bile transporter mRNAs quantified (Mrp1, Mrp2, Mrp3, Mrp5, Ntcp, Bsep, Oatp2), only 3 showed significant differences (Mrp2, Ntcp, Oatp2) (Fig 2). Mrp2 mRNA, as previously reported, had been elevated in homozygous affected (Npc1/) mice [Erickson, et al, 2005], more so with a high-fat diet. There was a slight, but significant, decrease in Mrp2 mRNA in Npc1+/+ females fed a high-fat diet compared to Npc1+/+ females fed a regular diet. When compared to males on a regular diet, Mrp2 mRNA was also decreased in Npc1+/+ females fed a high-fat diet but the difference was not significant (p = 0.08) (Fig 2A). At the protein level, Mrp2 was markedly decreased in females of either genotype fed the high fat diet (Fig 3A). Moreover, Mrp2 protein was decreased in both female genotypes fed the high fat diet and Npc1+/ females fed a regular diet as compared to males on a regular diet (Fig 3A). Ntcp mRNA had also been elevated in Npc1/ on regular diet and decreased with the high-fat diet in Npc1/ females [Erickson, et al, 2005]. In the current work, the Npc1+/+ females fed a regular diet showed a significant increase in Ntcp mRNA compared to all studied groups. There was a significant decrease in Ntcp mRNA in both Npc1+/+ and Npc1+/ females when fed the high-fat diet (Fig 2B). Additionally, Ntcp protein levels were remarkably decreased in females of both genotypes when fed the high fat diet, and Ntcp protein levels were greatly diminished in all females compared to males (Fig 3B). Oatp2, which had the same levels of mRNA expression as Ntcp in Npc1/ [Erickson, et al, 2005], showed no differences in the pattern of gene expression between the heterozygous and homozygous wild-typed females on either diet while all four female groups showed a significant increase in mRNA compared to regular diet fed Npc1+/+ males (Fig 2C). Similar to Ntcp, Oatp2 protein levels were markedly decreased in females of both genotypes fed the high fat diet (Fig 3C). As well, females of both genotypes fed the high fat diet exhibited decreased protein levels of Oatp2 compared to males (Fig 3C).

Fig 2
Relative amounts of Mrp2, Ntcp, and Oatp2 mRNA. Messenger RNA abundance for liver transporters Mrp2 (A) Ntcp (B) and Oatp2 (C) of female Npc1+/+ and Npc1+/− fed either regular or high-fat diet along with regular diet fed Npc1+/+ male are presented. ...
Fig 3
Relative amounts of Mrp2, Ntcp, and Oatp2 protein as determined by Western blotting. Relative protein abundance compared to Gapdh in livers for Mrp2 (A) Ntcp (B) and OatP2 (C) of female Npc1+/+ and Npc1+/− fed either regular or high-fat diet along ...

3.3 Caveolin 1 and Ptrf levels in liver and adipose tissue

The studies performed more than a decade ago, and for which laboratory records are no longer available, which reported increased caveolin 1 levels in Npc1+/ mouse liver (Introduction) did not state the sex of the mice. They were likely to be males since males are not affected by estrous cycle which alters many aspects of metabolism. We now find that caveolin 1 is significantly elevated in Npc1+/ male liver, but not Npc1+/ female liver when compared to the Npc1+/+ same sex animals fed the same regular diet (Fig 4A). Caveolin 1 levels in female liver for both Npc1+/ and Npc1+/+ are comparable to male Npc1+/ levels. On the other hand, the female Npc1+/ caveolin 1 levels in white adipose tissue are significantly higher when compared to those of Npc1+/+ females. We did not detect any difference between Npc1+/+ and Npc1+/ males for white adipose tissue levels of caveolin 1 (Fig 4B). Polymerase 1 and transcript release factor (Ptrf, another caveolae marker) levels were not different between the sexes or genotypes in white adipose tissue (levels were below our limits of detection in liver) (Fig 4C).

Fig 4
Relative amounts of caveolin-1 protein in 3 week old male and female Npc1+/+ and Npc1+/− liver and caveolin-1 and Ptrf protein in white adipose tissue. The average amounts (mean ± SE) of caveolin-1 protein in liver (A), caveolin-1 protein ...

3.4 Caveolar numbers

As seen in Fig 5, caveolae are much more abundant in adipose tissue than in liver. Thus caveolar numbers and diameters were compared for the adipose tissue in which elevated levels of caveolin 1 were found in heterozygotes, i.e. in female white adipose tissue. There were no significant differences in number of caveolae or their diameter (Table 2) but the numbers were highly variable.

Fig 5
Electron microscopy of caveolae in liver (A, B) and gonadal white adipose tissue (C, D). A representative image of liver caveolae of Npc1+/+ (A) and Npc1+/ mice fed a regular diet (B), and white adipose tissue of Npc1+/+ (C) and Npc1+/ ...

4. Discussion

4.1 NPC1 and obesity

We originally studied the effects of a high fat diet on homozygous recessive Npc1/ mice which have liver disease as well as the neurodegenerative disease. This work was stimulated since some cases of NPC1 may be missed because of death from cholestatic liver disease before development of neurological disease [Kelly, et al, 1993]. In fact, one study concluded that NPC is the second most common genetic cause of liver disease in infancy in the United Kingdom [Mieli-Vergani, et al, 1991]. A recent survey found that NPC explained 27% of idiopathic neonatal cholestasis and 8% of all infants evaluated for cholestasis [Yerushalmi, et al 2002].

Our interest in the effects of heterozygosity for Npc1 on obesity followed the findings of a Genome Wide Association Study (GWAS) implicating variation in NPC1 with early-onset (<6 yrs.) and morbid (BMI >40) adult obesity [Meyre, et al, 2009]. 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 non-obese individuals. The positively associated non-synonymous single nucleotide polymorphism (SNP) variant (rs1805081) results in the replacement of an arginine for a histidine at amino acid 215 (H215R). Histidine 215 is the more common allele and is the allele associated with lower risks of early onset morbid obesity [Meyre, et al, 2009].

Two other SNPs in NPC1 were also correlated with this finding. Ikonen’s group [Uronen, et al, 2010] found that intronic and 3’ SNPs in NPC1 significantly influenced serum triglyceride levels. Previous data had shown that heterozygous NPC1 mice had a significant accumulation of triglyceride [Garver, et al 2007]. This was a somewhat surprising finding since the homozygous recessive mice with severe liver disease had normal triglyceride levels [Garver, et al, 2007] while others reported lower than normal levels [Uronen,et al, 2003]. We had not previously characterized weight gain and general adiposity in heterozygotes. When we did, we found that male Npc1+/ mice, which express one-half the normal amount of functional Npc1 protein, had no significant difference in growth rate or weight gain when fed a low-fat diet but had a significantly increased growth rate and weight gain when fed a high fat diet [Jelinek, et al, 2010]. The mice also had a significantly increased liver weight and epididymal pad adipose weight. These studies were performed on the Balb/cJ genetic background. We now show similar results for female mice which may have differed because there are many sex effects on weight gain [Louet, et al, 2004; Gao & Horvath, 2008].

We have previously studied the liver disease in the Npc1/ mice. Serum alanine aminotransferase was elevated in Npc1/ mice on a regular diet and became markedly elevated with a high fat diet [Erickson, et al, 2005]. Non-zone dependent diffuse fibrosis was found in mice surviving the high fat diet (18% fat, 1% cholesterol) suggestive of non-alcoholic fatty liver disease (NAFLD). Bile duct function is altered in NAFLD [Fisher, et al, 2009] and was altered in Npc1/ with increases in gamma-glutaryl transferase and alterations in bile transporters: Mrps 1–5 were elevated in Npc1/ liver and became more elevated with the high fat diet; Ntcp, Bsep, and Oatp2 were also elevated in Npc1/ liver but were suppressed by the high fat diet [Erickson, et al, 2005]. This previous work has been confirmed and amplified. The cholesterol accumulation with high fat diet in Npc1/ mice correlated with cell death [Beltroy, et al, 2007] and could be partially prevented with ezetimibe therapy [Beltroy, et al, 2007; Zheng, et al, 2008].

4.3 NPC1 and NAFLD

NAFLD is rapidly becoming a major health problem [Barshop, et al, 2008], and the liver disease in Npc1/ mice provides a partial model. Since bile duct transporters were altered in the homozygous recessive mice and are known to be altered in NAFLD, we studied them in the heterozygotes. We now find that Mrp2 mRNA showed significant differences between high fat versus normal diet in Npc1+/+ females (p<0.05) but were not as greatly elevated as in Npc1/ on a high fat diet. However, the protein levels determined by Western blots were markedly depressed in the females of both genotypes fed the high fat diet. The mRNA for Ntcp, which transports bile acids from portal blood into hepatocytes, was also elevated in Npc1/ on a regular diet [Erickson, et al, 2005] but decreased with high fat diet as it does in female Npc1+/+ liver. In our current work, high fat diet caused a marked decrease in the protein level of this transporter in females of both genotypes. Oatp2 mRNA had shown a pattern similar to that of Ntcp in Npc1/ on a high fat diet [Erickson, et al, 2005] while it is now found to be consistently elevated in females compared to males regardless of the diet. At the protein level, it, too, was markedly lowered by the high fat diet treatment. Overall, the changes do not suggest a consistent pattern related to the increased weight gain of Npc1+/ compared to Npc1+/+ mice since both genotypes shared small changes in mRNA levels and large changes in protein levels induced by the high fat diet. The effects of similar changes in transporter function with a high fat diet (non-alcoholic fatty liver disease model) on bromosulfophthalein disposition have been well established in animal models [Fisher, et al, 2009].

There are many post-translational modifications of bile transporters: Ntcp by phosphorylation [Anwer, et al, 2005] and ubiquitanation, which leads to proteosomal degradation [Kuhlkamp, et al, 2005]; Mrp2 by SUMOlyation, which confers stability to the protein [Minami, et al, 2009]; and Mrp2 by glycosylation which, if incomplete, prevents routing of the protein to the membrane and may lead to degradation [Zhang, et al, 2005]. Genetic translational regulation is only well studied for Mrp2 [Zhang, et al, 2007; Zhang, et al, 2010]. This was first found following PXR stimulation with pregnenolone-16alpha-carbonitrile (PCN [Johnson, et al, 2002]) and was found to be due to de novo protein synthesis without changes in mRNA levels [Jones, et al, 2005].

4.4 NPC1, caveolin 1, and possible links to obesity

Using the BALB/c murine model for NPC1 disease, we previously found that the expression of caveolin 1 in total liver homogenates from Npc1+/ and Npc1/ animals was altered [Garver, et al 1997a]. Immunoblot analysis of liver homogenates from Npc1+/ mice revealed that caveolin 1 was significantly (p<0.005) elevated, 4.9 fold, compared to normal mice. Total liver homogenates from Npc1/ mice also had a significant (p<0.05) increase in caveolin 1 expression, 1.6 fold, compared to normal Npc1+/+ mice. Immunohistochemical staining of liver cross-sections revealed that the increased caveolin 1 was localized to sinusoidal endothelial cells in heterozygous mice [Garver, et al, 1997a]. The Triton insoluble floating fraction (TIFF) i.e. lipid rafts, was isolated using liver from each genotype and analyzed for caveolin 1 expression. Caveolin 1 in the TIFF from Npc1+/ mice was significantly (p<0.01) elevated, 1.8 fold, compared to Npc1+/+ and Npc1/ animals; while Npc1+/+ and Npc1/ animals, were not significantly different from each other.

We also studied caveolin 1 levels in cultured human skin fibroblasts [Garver, et al, 1997b]. Caveolin 1 in heterozygous fibroblasts was significantly elevated, as much as 10-fold compared to caveolin 1 in normal and homozygous affected fibroblasts. Homozygous NPC fibroblasts expressed caveolin 1 levels similar to those in normal fibroblasts, while the expression of caveolin 1 in homozygous Niemann-Pick type D (NPD, a milder form due to a hypomorphic mutation in NPC1) fibroblasts was slightly elevated. Northern analyses indicated that normal fibroblasts and NPC heterozygous fibroblasts have similar amounts of caveolin 1 mRNA, while NPC homozygous fibroblasts have significantly less caveolin 1 mRNA. In contrast, heterozygous and homozygous NPD fibroblasts exhibit increased levels of caveolin 1 mRNA.

Thus, because caveolae have a major role on fat metabolism (see Introduction) we tested the hypothesis that the increase in caveolin 1 would be reflected in an increased number of caveolae. Although the variation was extremely large, there were no significant differences in the numbers of caveolae in the Npc1+/− and Npc1+/+ female white adipose tissue. There was also no difference in Ptrf levels which are another marker for caveolae. We have not studied muscle where similar metabolic changes are found in the sarcolemma, in which caveolin 3 is the form of caveolin predominantly associated with caveolae [Parton, et al,1997].

In conclusion, we have shown that reduced Npc1 levels are associated with increased adiposity in both sexes when the mice are fed a high fat diet. The adiposity is associated with changes in liver bile transporters but not with detectable changes in caveolar number or Ptrf levels.

Highlights

  • Female Npc1 heterozygotes, but not wild-type, mice become obese on a high fat diet
  • High fat diet up-regulated some bile acid transporters in female Npc1 heterozygotes
  • Changes in caveolar proteins or numbers did not explain the Npc1 obesity effects

Acknowledgments

Support: NIH 5RO1 EB000343-05 (T Trouard, PI); NIH DK068039 (N. Cherrington, PI); and the Holsclaw Family Professorship of Human Genetics and Inherited Disease (RPE)

List of abbreviations

BSEP
bile salt export pump
ECL
enhanced chemiluminescence
GLUT4
glucose transporter 4
KO
knockout
Mrp
multidrug resistant protein
NAFLD
non-alcoholic fatty liver disease
Npc1
mouse Niemann-Pick C1 gene or protein
NPC1
Niemann-Pick C1 type disease
NPD
a milder form of NPC1
Ntcp
Na+-taurocholate co-transporter polypeptide
Oat
organic ion transport protein
PAGE
polyacrylamide gel electrophoresis
PCN
pregnenolone-16-alpha carbonitrile
Ptrf
polymerase and transcript release factor
PXR
pregnane X receptor
SDS
sodium dodecyl sulfate
SNP
single nucleotide polymorphism
TIFF
triton incoluble floating fraction

Footnotes

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References

1. Anwer MS, Gillin H, Mukhopadhyay S, Balasubramaniyan N, Suchy FJ, Ananthanarayanan M. Dephosphorylation of Ser-226 facilitates plasma membrane retention of Ntcp. J Biol Chem. 2005;280:33687–33692. [PubMed]
2. Barshop NJ, Sirlin CB, Schwimmer JB, Lavine JE. Review article: epidemiology, pathogenesis and potential treatments of paediatric non-alcoholic fatty liver disease. Aliment Pharmacol Ther. 2008;28:13–24. [PubMed]
3. Beltroy EP, Liu B, Dietschy JM, Turley SD. Lysosomal unesterified cholesterol content correlates with liver cell death in murine Niemann-Pick C disease. J Lipid Res. 2007;48:869–81. [PubMed]
4. Carstea ED, Morris JA, Coleman KG, Loftus SK, Zhang D, Cummings C, Gu J, Rosenfeld MA, Pavan WJ, Kirzman DB, Nagle J, Polymeropoulos MH, Sturley SL, Ioannou YA, Higgins ME, Comly M, Cooney A, Brown A, Kaneski CR, Blanchette-Mackie EJ, Dwyer NK, Neufeld EB, Chang TY, Liscum L, Strauss JF, Ohno K, Zeigler M, Carmi R, Sokol J, Markie D, O’Neill RR, vanDiggelen OP, Elleder M, Patterson MC, Brady RO, Vanier MT, Pentchev PG, Tagle DA. Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis. Science. 1997;277:228–31. [PubMed]
5. Catalan V, Gomez-Ambrosi J, Rodriguez A, Silva C, Rotellar F, Gil MJ, Cienfuegos JA, Salvador J, Fruhbeck G. Expression of caveolin-1 in human adipose tissue is upregulated in obesity and obesity-associated type 2 diabetes mellitus and related to inflammation. Clin Endocrinol. 2008;68:213–219. [PubMed]
6. Cohen AW, Razani B, Wang XB, Combs TP, Williams TM, Scherer PE, Lisanti MP. Caveolin-1-deficient mice show insulin resistance and defective insulin receptor protein expression in adipose tissue. Am J Physiol Cell Physiol. 2003;285:C222–35. [PubMed]
7. Erickson RP, Bhattacharyya A, Hunter RJ, Heidenreich RA, Cherrington NJ. Liver disease with altered bile transport in Niemann-Pick C mice on a high fat 1% cholesterol diet. Am J Physiol. 2005;289:G300–G307. [PubMed]
8. Fisher CD, Lickteig AJ, Augustine LM, Elferink RPJO, Besselsen DG, Erickson RP, Cherrington NJ. Experimental non-alcoholic fatty liver disease results in decreased hepatic uptake transporter expression and function in rats. Eur J Pharmacol. 2009;613:119–127. [PMC free article] [PubMed]
9. Gao Q, Horvath TL. Cross-talk between estrogen and leptin signaling in the hypothalamus. Amer J Physiol-Endocrin and Metab. 2008;294:E817–826. [PubMed]
10. Garver WS, Erickson RP, Wilson JM, Colton TL, Hossain GS, Kozloski MA, Heidenreich RA. Altered expression of caveolin 1 and increased cholesterol in detergent insoluble membrane fractions from liver in mice with Niemann-Pick disease type C. Biochim Biophys Acta. 1997a;361:272–80. [PubMed]
11. 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]
12. Garver WS, Jelinek D, Oyarzo JN, Flynn J, Zuckerman M, Krishman K, Chung BH, Heidenreich RA. Characterization of liver disease and lipid metabolism in the Niemann-Pick C1 mouse. J Cell Biochem. 2007;101:498–516. [PubMed]
13. Garver WS, Ssu-Cheng J, Erickson RP, Greer WL, Byers DM, Heidenreich RA. Increased expression of caveolin 1 in heterozygous Niemann-Pick type II human fibroblasts. Biochem Biophys Res Comm. 1997b;36:189–93. [PubMed]
14. Gustavsson J, Parpal S, Karlsson M, Ramsing C, Thorn H, Borg M, Lindroth M, Peterson KH, Magnusson KE, Strâlfors P. Localization of the insulin receptor in caveolae of adipocyte plasma membrane. FASEB J. 1999;13:1961. [PubMed]
15. 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. [PubMed]
16. Jelinek D, Heidenreich RA, Erickson RP, Garver WS. Decreased Npc1 gene dosage in mice is associated with weight gain. Obesity. 2010;18:1457–9. [PMC free article] [PubMed]
17. Johnson DR, Klaassen CD. Regulation of rat multidrug resistance protein 2 by classes of prototypical microsomal enzyme inducers that activate distinct transcription pathways. Toxicol Sci. 2002;67:182–189. [PubMed]
18. Jones BR, Li W, Cao J, Hoffman TA, Gerk PM, Vore M. The role of protein synthesis and degradation in the post-transcriptional regulation of rat multidrug resistance-associated protein 2 (Mrp2, Abcc2) Mol Pharmacol. 2005;68:701–710. [PubMed]
19. Kelly DA, Portmann B, Mowat AP, Sherlock S, Lake BD. Niemann-Pick disease type C: diagnosis and outcome in children, with particular reference to liver disease. J Pediatr. 1993;123:242–7. [PubMed]
20. Kühlkamp T, Keitel V, Helmer A, Häussinger D, Kubitz R. Degradation of the sodium taurocholate cotransporting polypeptide (NTCP) by the ubiquitinproteasome system. Biol Chem. 2005;386:1065–1074. [PubMed]
21. Loftus SK, Morris JA, Carstea ED, Gu JZ, Cummings C, Brown A, Ellison J, Ohno K, Rosenfeld MA, Tagle DA, Pentchev PG, Pavan WJ. Murine model of Niemann-Pick C disease: mutation in a cholesterol homeostasis gene. Science. 1997;277:232–5. [PubMed]
22. Louet JF, LeMan C, Mauvais-Jarvis F. Antidiabetic Actions of Estrogen: Insight from Human and Genetic Mouse Models. Curr Atheroscler Reports. 2004;6:180–185. [PubMed]
23. Meyre D, Delplanque J, Chèvre JC, Lecoeur C, Lobbens S, Gallina S, Durand E, Vatin V, Degraeve F, Proença C, Gaget S, Körner A, Kovacs P, Kiess W, Tichet J, Marre M, Hartikainen AL, Horber F, Potoczna N, Hercberg S, Levy-Marchal C, Pattou F, Heude B, Tauber M, McCarthy MI, Blakemore AIF, Montpetit A, Polychronakos C, Weill J, Coin LJM, Asher J, Elliott P, Järvelin MR, Visvikis-Siest S, Balkau B, Sladek R, Balding D, Walley A, Dina C, Froguel P. Genome-wide association study for early-onset and morbid adult obesity identifies three new risk loci in European populations. Nat Genet. 2009;41:157–159. [PubMed]
24. Mieli-Vergani G, Howard ER, Mowat AP. Liver disease in infancy: a 20-year perspective. Gut. 1991;(Suppl):S123–S128. [PMC free article] [PubMed]
25. Minami S, Ito K, Honma M, Ikebuchi Y, Anzai N, Kanai Y, Nishida T, Tsukita S, Sekine S, Horie T, et al. Posttranslational regulation of Abcc2 expression by SUMOylation system. Am J Physiol Gastrointest Liver Physiol. 2009;296:G406–G413. [PubMed]
26. Neufeld EB, Wastney M, Patel S, Suresh S, Cooney AM, Dwyer NK, Roff CF, Ohno K, Morris JA, Carstea ED, Incardona JP, Strauss JF, Vanier MT, Patterson MC, Brady RO, Pentchev PG, Blanchette-Mackie EJ. The Niemann-Pick C1 protein resides in a vesicular compartment linked to retrograde transport of multiple lysosomal cargo. J Biol Chem. 1999;274:9627–35. [PubMed]
27. Ortegren U, Yin L, Ost A, Karlsson H, Nystrom FH, Strålfors P. Separation and characterization of caveolae subclasses in the plasma membrane of primary adipocytes; segregation of specific proteins and functions. FEBS J. 2006;273:3381–92. [PubMed]
28. Parton RG, Way M, Zorzi N, Stang E. Caveolin-3 associates with developing T-tubules during muscle differentiation. J Cell Biol. 1997;136:137–54. [PMC free article] [PubMed]
29. Patterson MC, Vanier MT, Suzuki K, Morris JA, Carstea ED, Neufeld EB, Blanchette-Mackie EJ, Pentchev PG. Niemann-Pick disease type C: a lipid trafficking disorder. In: Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B, Kinzler KW, Vogelstein B, editors. The metabolic and molecular bases of inherited disease. 8. McGraw-Hill; New York: 2001. pp. 3611–34.
30. Rauch MC, Ocampo ME, Bohle J, Amthauer R, Yáñez AJ, Rodríguez-Gil JE, Slebe JC, Reyes JG, Concha II. Hexose transporters GLUT1 and GLUT3 are colocalized with hexokinase I in caveolae microdomains of rat spermatogenic cells. J Cell Physiol. 2006;207:397–406. [PubMed]
31. Razani B, Combs TP, Wang XB, Frank PG, Park DS, Russell RG, Li M, Tang B, Jelicks LA, Scherer PE, Lisanti MP. Caveolin-1-deficient mice are lean, resistant to diet-induced obesity, and show hypertriglyceridemia with adipocyte abnormalities. J Biol Chem. 2002;277:8635–47. [PubMed]
32. Ring, Le Lay S, Pohl J, Verkade P, Stremmel W. Caveolin 1 is required for fatty acid translocase (FAT/CD36) localization and function at the plasma membrane of mouse embryonic fibroblasts. Biochim Biophys Acta. 2006;1761:416–23. [PubMed]
33. Scherer PE, Lisanti MP, Baldini G, Sargiacomo M, Mastick CC, Lodish HF. Induction of caveolin during adipogenesis and association of GLUT4 with caveolin-rich vesicles. J Cell Biol. 1994;127:1233–43. [PMC free article] [PubMed]
34. Trigatti BL, Anderson RG, Gerber GE. Identification of caveolin 1 as a fatty acid binding protein. Biochem Biophys Res Comm. 1999;255:34–9. [PubMed]
35. Uronen RL, Lundmark P, Orho-Melander M, Jauhiainen M, Larsson K, Siegbahn A, Wallentin L, Zethelius B, Melander O, Syvanen AC, Ikonen E. Niemann-Pick C1 modulates hepatic triglyceride metabolism and its genetic variation contributes to serum triglyceride levels. Artericler Thromb Vasc Biol. 2010;30:1614–20. [PubMed]
36. Yerushalmi B, Sokol RJ, Narkewicz MR, Smith D, Ashmead JW, Wenger DA. Niemann-Pick disease type C in neonatal cholestasis at a North American center. J Pediatr Gastroenterol Nutr. 2002;35:44–50. [PubMed]
37. Zhang P, Tian X, Chandra P, Brouwer KL. Role of glycosylation in trafficking of Mrp2 in sandwich-cultured rat hepatocytes. Mol Pharmacol. 2005;67:1334–1341. [PubMed]
38. Zhang Y, Li W, Vore M. Translational regulation of rat multidrug resistance-associated protein 2 expression is mediated by upstream open reading frames in the 5 untranslated region. Mol Pharmacol. 2007;71:377–383. [PubMed]
39. Zhang Y, Zhao T, Li W, Vore M. The 5-untranslated region of multidrug resistance associated protein 2 (MRP2; ABCC2) regulates downstream open reading frame expression through translational regulation. Mol Pharmacol. 2010;77:237–246. [PMC free article] [PubMed]
40. Zheng S, Hoos L, Cook J, Tetzloff G, Davies H, Jr, van Heek M, Hwa JJ. Ezetimibe improves high fat and cholesterol diet-induced non-alcoholic fatty liver disease in mice. Eur J Pharmacol. 2008;584:118–24. [PubMed]
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