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author:("ree, Karen")
1.  Cell-autonomous sex determination outside of the gonad 
The classic model of sex determination in mammals states that the sex of the individual is determined by the type of gonad that develops, which in turn determines the gonadal hormonal milieu that creates sex differences outside of the gonads. However, XX and XY cells are intrinsically different because of the cell-autonomous sex-biasing action of X and Y genes. Recent studies of mice, in which sex chromosome complement is independent of gonadal sex, reveal that sex chromosome complement has strong effects contributing to sex differences in phenotypes such as metabolism. Adult mice with two X chromosomes (relative to mice with one X chromosome) show dramatically greater increases in body weight and adiposity after gonadectomy, irrespective of their gonadal sex. When fed a high fat diet, XX mice develop striking hyperinsulinemia and fatty liver, relative to XY mice. The sex chromosome effects are modulated by the presence of gonadal hormones, indicating an interaction of the sex-biasing effects of gonadal hormones and sex chromosome genes. Other cell-autonomous sex chromosome effects are detected in mice in many phenotypes. Birds (relative to eutherian mammals) are expected to show more widespread cell-autonomous sex determination in non-gonadal tissues, because of ineffective sex chromosome dosage compensation mechanisms.
doi:10.1002/dvdy.23936
PMCID: PMC3672066  PMID: 23361913
sex chromosome; sex determination; X chromosome; Y chromosome; Z chromosome; W chromosome; sexual differentiation; androgens; estrogens
2.  Follistatin promotes adipocyte differentiation, browning, and energy metabolism[S] 
Journal of Lipid Research  2014;55(3):375-384.
Follistatin (Fst) functions to bind and neutralize the activity of members of the transforming growth factor-β superfamily. Fst has a well-established role in skeletal muscle, but we detected significant Fst expression levels in interscapular brown and subcutaneous white adipose tissue, and further investigated its role in adipocyte biology. Fst expression was induced during adipogenic differentiation of mouse brown preadipocytes and mouse embryonic fibroblasts (MEFs) as well as in cold-induced brown adipose tissue from mice. In differentiated MEFs from Fst KO mice, the induction of brown adipocyte proteins including uncoupling protein 1, PR domain containing 16, and PPAR gamma coactivator-1α was attenuated, but could be rescued by treatment with recombinant FST. Furthermore, Fst enhanced thermogenic gene expression in differentiated mouse brown adipocytes and MEF cultures from both WT and Fst KO groups, suggesting that Fst produced by adipocytes may act in a paracrine manner. Our microarray gene expression profiling of WT and Fst KO MEFs during adipogenic differentiation identified several genes implicated in lipid and energy metabolism that were significantly downregulated in Fst KO MEFs. Furthermore, Fst treatment significantly increases cellular respiration in Fst-deficient cells. Our results implicate a novel role of Fst in the induction of brown adipocyte character and regulation of energy metabolism.
doi:10.1194/jlr.M039719
PMCID: PMC3934723  PMID: 24443561
mouse embryonic fibroblast; myostatin; brown fat; energy expenditure; uncoupling protein 1; mitochondria
3.  Skeletal muscle Nur77 expression enhances oxidative metabolism and substrate utilization[S] 
Journal of Lipid Research  2012;53(12):2610-2619.
Mitochondrial dysfunction has been implicated in the pathogenesis of type 2 diabetes. Identifying novel regulators of mitochondrial bioenergetics will broaden our understanding of regulatory checkpoints that coordinate complex metabolic pathways. We previously showed that Nur77, an orphan nuclear receptor of the NR4A family, regulates the expression of genes linked to glucose utilization. Here we demonstrate that expression of Nur77 in skeletal muscle also enhances mitochondrial function. We generated MCK-Nur77 transgenic mice that express wild-type Nur77 specifically in skeletal muscle. Nur77-overexpressing muscle had increased abundance of oxidative muscle fibers and mitochondrial DNA content. Transgenic muscle also exhibited enhanced oxidative metabolism, suggestive of increased mitochondrial activity. Metabolomic analysis confirmed that Nur77 transgenic muscle favored fatty acid oxidation over glucose oxidation, mimicking the metabolic profile of fasting. Nur77 expression also improved the intrinsic respiratory capacity of isolated mitochondria, likely due to the increased abundance of complex I of the electron transport chain. These changes in mitochondrial metabolism translated to improved muscle contractile function ex vivo and improved cold tolerance in vivo. Our studies outline a novel role for Nur77 in the regulation of oxidative metabolism and mitochondrial activity in skeletal muscle.
doi:10.1194/jlr.M029355
PMCID: PMC3494265  PMID: 23028113
Nr4a; nuclear receptor; mitochondria
4.  Lipin-1 and lipin-3 together determine adiposity in vivo☆ 
Molecular Metabolism  2013;3(2):145-154.
The lipin protein family of phosphatidate phosphatases has an established role in triacylglycerol synthesis and storage. Physiological roles for lipin-1 and lipin-2 have been identified, but the role of lipin-3 has remained mysterious. Using lipin single- and double-knockout models we identified a cooperative relationship between lipin-3 and lipin-1 that influences adipogenesis in vitro and adiposity in vivo. Furthermore, natural genetic variations in Lpin1 and Lpin3 expression levels across 100 mouse strains correlate with adiposity. Analysis of PAP activity in additional metabolic tissues from lipin single- and double-knockout mice also revealed roles for lipin-1 and lipin-3 in spleen, kidney, and liver, for lipin-1 alone in heart and skeletal muscle, and for lipin-1 and lipin-2 in lung and brain. Our findings establish that lipin-1 and lipin-3 cooperate in vivo to determine adipose tissue PAP activity and adiposity, and may have implications in understanding the protection of lipin-1-deficient humans from overt lipodystrophy.
doi:10.1016/j.molmet.2013.11.008
PMCID: PMC3953701  PMID: 24634820
Gene family; Knockout mouse; Adipogenesis; Triacylglycerol; Glycerolipid biosynthesis
5.  The sterol regulatory element binding proteins are essential for the metabolic programming of effector T cells and adaptive immunity 
Nature immunology  2013;14(5):489-499.
Newly activated CD8+ T cells reprogram their metabolism to meet the extraordinary biosynthetic demands of clonal expansion; however, the signals mediating metabolic reprogramming remain poorly defined. Herein, we demonstrate an essential role for sterol regulatory element binding proteins (SREBPs) in the acquisition of effector cell metabolism. Without SREBP signaling, CD8+ T cells are unable to blast, resulting in markedly attenuated clonal expansion during viral infection. Mechanistic studies indicate that SREBPs are essential to meet the heightened lipid requirements of membrane synthesis during blastogenesis. SREBPs are dispensable for homeostatic proliferation, indicating a context-specific requirement for SREBPs in effector responses. These studies provide insights into the molecular signals underlying metabolic reprogramming of CD8+ T cells during the transition from quiescence to activation.
doi:10.1038/ni.2570
PMCID: PMC3652626  PMID: 23563690
SREBP; LCMV; lipids; CD8+ T cell; metabolism; proliferation
6.  Adipose subtype–selective recruitment of TLE3 or Prdm16 by PPARγ specifies lipid-storage versus thermogenic gene programs 
Cell metabolism  2013;17(3):423-435.
Transcriptional effectors of white adipocyte-selective gene expression have not been described. Here we show that TLE3 is a white-selective cofactor that acts reciprocally with the brown-selective cofactor Prdm16 to specify lipid storage and thermogenic gene programs. Occupancy of TLE3 and Prdm16 on certain promoters is mutually exclusive, due to the ability of TLE3 to disrupt the physical interaction between Prdm16 and PPARγ. When expressed at elevated levels in brown fat, TLE3 counters Prdm16, suppressing brown-selective genes and inducing white-selective genes, resulting in impaired fatty acid oxidation and thermogenesis. Conversely, mice lacking TLE3 in adipose tissue show enhanced thermogenesis in inguinal white adipose depots and are protected from age-dependent deterioration of brown adipose tissue function. Our results suggest that the establishment of distinct adipocyte phenotypes with different capacities for thermogenesis and lipid storage is accomplished in part through the cell type–selective recruitment of TLE3 or Prdm16 to key adipocyte target genes.
doi:10.1016/j.cmet.2013.01.016
PMCID: PMC3626567  PMID: 23473036
7.  Lipins: Multifunctional Lipid Metabolism Proteins 
Annual review of nutrition  2010;30:257-272.
The lipin proteins are evolutionarily conserved proteins with roles in lipid metabolism and disease. There are three lipin protein family members in mammals and one or two orthologs in plants, invertebrates, and single-celled eukaryotes. Studies in yeast and mouse led to the identification of two distinct molecular functions of lipin proteins. Lipin proteins have phosphatidate phosphatase activity and catalyze the formation of diacylglycerol in the glycerol-3-phosphate pathway, implicating them in the regulation of triglyceride and phospholipid biosynthesis. Mammalian lipin proteins also possess transcriptional coactivator activity and have been implicated in the regulation of metabolic gene expression. Here we review key findings in the field that demonstrate roles for lipin family members in metabolic homeostasis and in rare human diseases, and we examine evidence implicating genetic variations in lipin genes in common metabolic dysregulation such as obesity, hyperinsulinemia, hypertension, and type 2 diabetes.
doi:10.1146/annurev.nutr.012809.104729
PMCID: PMC3738581  PMID: 20645851
triglyceride; obesity; insulin resistance; phosphatidate phosphatase; transcriptional coactivator
8.  The Sex Chromosome Trisomy mouse model of XXY and XYY: metabolism and motor performance 
Background
Klinefelter syndrome (KS), caused by XXY karyotype, is characterized by low testosterone, infertility, cognitive deficits, and increased prevalence of health problems including obesity and diabetes. It has been difficult to separate direct genetic effects from hormonal effects in human studies or in mouse models of KS because low testosterone levels are confounded with sex chromosome complement.
Methods
In this study, we present the Sex Chromosome Trisomy (SCT) mouse model that produces XXY, XYY, XY, and XX mice in the same litters, each genotype with either testes or ovaries. The independence of sex chromosome complement and gonadal type allows for improved recognition of sex chromosome effects that are not dependent on levels of gonadal hormones. All mice were gonadectomized and treated with testosterone for 3 weeks. Body weight, body composition, and motor function were measured.
Results
Before hormonal manipulation, XXY mice of both sexes had significantly greater body weight and relative fat mass compared to XY mice. After gonadectomy and testosterone replacement, XXY mice (both sexes) still had significantly greater body weight and relative fat mass, but less relative lean mass compared to XY mice. Liver, gonadal fat pad, and inguinal fat pad weights were also higher in XXY mice, independent of gonadal sex. In several of these measures, XX mice also differed from XY mice, and gonadal males and females differed significantly on almost every metabolic measure. The sex chromosome effects (except for testis size) were also seen in gonadally female mice before and after ovariectomy and testosterone treatment, indicating that they do not reflect group differences in levels of testicular secretions. XYY mice were similar to XY mice on body weight and metabolic variables but performed worse on motor tasks compared to other groups.
Conclusions
We find that the new SCT mouse model for XXY and XYY recapitulates features found in humans with these aneuploidies. We illustrate that this model has significant promise for unveiling the role of genetic effects compared to hormonal effects in these syndromes, because many phenotypes are different in XXY vs. XY gonadal female mice which have never been exposed to testicular secretions.
doi:10.1186/2042-6410-4-15
PMCID: PMC3751353  PMID: 23926958
Klinefelter; Sex chromosome trisomy; XXY; XYY; Mouse; X chromosome; Y chromosome; Body weight; Obesity
9.  PPM1l encodes an inositol requiring-protein 1 (IRE1) specific phosphatase that regulates the functional outcome of the ER stress response★ 
Molecular Metabolism  2013;2(4):405-416.
The protein phosphatase 1-like gene (PPM1l) was identified as causal gene for obesity and metabolic abnormalities in mice. However, the underlying mechanisms were unknown. In this report, we find PPM1l encodes an endoplasmic reticulum (ER) membrane targeted protein phosphatase (PP2Ce) and has specific activity to basal and ER stress induced auto-phosphorylation of Inositol-REquiring protein-1 (IRE1). PP2Ce inactivation resulted in elevated IRE1 phosphorylation and higher expression of XBP-1, CHOP, and BiP at basal. However, ER stress stimulated XBP-1 and BiP induction was blunted while CHOP induction was further enhanced in PP2Ce null cells. PP2Ce protein levels are significantly induced during adipogenesis in vitro and are necessary for normal adipocyte maturation. Finally, we provide evidence that common genetic variation of PPM11 gene is significantly associated with human lipid profile. Therefore, PPM1l mediated IRE1 regulation and downstream ER stress signaling is a plausible molecular basis for its role in metabolic regulation and disorder.
Graphical abstract
doi:10.1016/j.molmet.2013.07.005
PMCID: PMC3854994  PMID: 24327956
IRE1; PPM1l; Protein phosphatase; ER stress; Adipogenesis
10.  Metabolic impact of sex chromosomes 
Adipocyte  2013;2(2):74-79.
Obesity and associated metabolic diseases are sexually dimorphic. To provide better diagnosis and treatment for both sexes, it is of interest to identify the factors that underlie male/female differences in obesity. Traditionally, sexual dimorphism has been attributed to effects of gonadal hormones, which influence numerous metabolic processes. However, the XX/XY sex chromosome complement is an additional factor that may play a role. Recent data using the four core genotypes mouse model have revealed that sex chromosome complement—independently from gonadal sex—plays a role in adiposity, feeding behavior, fatty liver and glucose homeostasis. Potential mechanisms for the effects of sex chromosome complement include differential gene dosage from X chromosome genes that escape inactivation, and distinct genomic imprints on X chromosomes inherited from maternal or paternal parents. Here we review recent data in mice and humans concerning the potential impact of sex chromosome complement on obesity and metabolic disease.
doi:10.4161/adip.23320
PMCID: PMC3661109  PMID: 23805402
metabolic disease; sex differences; obesity; food intake; fatty liver; circadian rhythm
11.  Hybrid mouse diversity panel: a panel of inbred mouse strains suitable for analysis of complex genetic traits 
We have developed an association-based approach using classical inbred strains of mice in which we correct for population structure, which is very extensive in mice, using an efficient mixed-model algorithm. Our approach includes inbred parental strains as well as recombinant inbred strains in order to capture loci with effect sizes typical of complex traits in mice (in the range of 5 % of total trait variance). Over the last few years, we have typed the hybrid mouse diversity panel (HMDP) strains for a variety of clinical traits as well as intermediate phenotypes and have shown that the HMDP has sufficient power to map genes for highly complex traits with resolution that is in most cases less than a megabase. In this essay, we review our experience with the HMDP, describe various ongoing projects, and discuss how the HMDP may fit into the larger picture of common diseases and different approaches.
doi:10.1007/s00335-012-9411-5
PMCID: PMC3586763  PMID: 22892838
12.  Reciprocal Metabolic Perturbations in the Adipose Tissue and Liver of GPIHBP1-deficient Mice 
Objective
Gpihbp1-deficient mice (Gpihbp1−/−) lack the ability to transport lipoprotein lipase to the capillary lumen, resulting in mislocalization of LPL within tissues, defective lipolysis of triglyceride-rich lipoproteins, and chylomicronemia. We asked whether GPIHBP1 deficiency and mislocalization of catalytically active LPL would alter the composition of triglycerides in adipose tissue or perturb the expression of lipid biosynthetic genes. We also asked whether perturbations in adipose tissue composition and gene expression, if they occur, would be accompanied by reciprocal metabolic changes in the liver.
Methods and Results
The chylomicronemia in Gpihbp1−/− mice was associated with reduced levels of essential fatty acids in adipose tissue triglycerides and increased expression of lipid biosynthetic genes. The liver exhibited the opposite changes—increased levels of essential fatty acids in triglycerides and reduced expression of lipid biosynthetic genes.
Conclusions
Defective lipolysis in Gpihbp1−/− mice causes reciprocal metabolic perturbations in adipose tissue and liver. In adipose tissue, the essential fatty acid content of triglycerides is reduced and lipid biosynthetic gene expression is increased, while the opposite changes occur in the liver.
doi:10.1161/ATVBAHA.111.241406
PMCID: PMC3281771  PMID: 22173228
lipoprotein lipase; hypertriglyceridemia; lipolysis; essential fatty acids; lipid biosynthetic genes
13.  ABCC6 Localizes to the Mitochondria-Associated Membrane 
Circulation research  2012;111(5):516-520.
Rationale
Mutations of the orphan transporter ABCC6 (ATP-binding cassette, subfamily C, member 6) cause the connective tissue disorder pseudoxanthoma elasticum. ABCC6 was thought to be located on the plasma membrane of liver and kidney cells.
Objective
Mouse systems genetics and bioinformatics suggested that ABCC6 deficiency affects mitochondrial gene expression. We therefore tested whether ABCC6 associates with mitochondria.
Methods and Results
We found ABCC6 in crude mitochondrial fractions and subsequently pinpointed its localization to the purified mitochondria-associated membrane fraction. Cell-surface biotinylation in hepatocytes confirmed that ABCC6 is intracellular. Abcc6-knockout mice demonstrated mitochondrial abnormalities and decreased respiration reserve capacity.
Conclusions
Our finding that ABCC6 localizes to the mitochondria-associated membrane has implications for its mechanism of action in normal and diseased states.
doi:10.1161/CIRCRESAHA.112.276667
PMCID: PMC3540978  PMID: 22811557
PXE; vascular calcification; ABCC6/MRP6; MAM; mitochondria; cardiovascular disease
14.  The Number of X Chromosomes Causes Sex Differences in Adiposity in Mice 
PLoS Genetics  2012;8(5):e1002709.
Sexual dimorphism in body weight, fat distribution, and metabolic disease has been attributed largely to differential effects of male and female gonadal hormones. Here, we report that the number of X chromosomes within cells also contributes to these sex differences. We employed a unique mouse model, known as the “four core genotypes,” to distinguish between effects of gonadal sex (testes or ovaries) and sex chromosomes (XX or XY). With this model, we produced gonadal male and female mice carrying XX or XY sex chromosome complements. Mice were gonadectomized to remove the acute effects of gonadal hormones and to uncover effects of sex chromosome complement on obesity. Mice with XX sex chromosomes (relative to XY), regardless of their type of gonad, had up to 2-fold increased adiposity and greater food intake during daylight hours, when mice are normally inactive. Mice with two X chromosomes also had accelerated weight gain on a high fat diet and developed fatty liver and elevated lipid and insulin levels. Further genetic studies with mice carrying XO and XXY chromosome complements revealed that the differences between XX and XY mice are attributable to dosage of the X chromosome, rather than effects of the Y chromosome. A subset of genes that escape X chromosome inactivation exhibited higher expression levels in adipose tissue and liver of XX compared to XY mice, and may contribute to the sex differences in obesity. Overall, our study is the first to identify sex chromosome complement, a factor distinguishing all male and female cells, as a cause of sex differences in obesity and metabolism.
Author Summary
Differences exist between men and women in the development of obesity and related metabolic diseases such as type 2 diabetes and cardiovascular disease. Previous studies have focused on the sex-biasing role of hormones produced by male and female gonads, but these cannot account fully for the sex differences in metabolism. We discovered that removal of the gonads uncovers an important genetic determinant of sex differences in obesity—the presence of XX or XY sex chromosomes. We used a novel mouse model to tease apart the effects of male and female gonads from the effects of XX or XY chromosomes. Mice with XX sex chromosomes (relative to XY), regardless of their type of gonad, had increased body fat and ate more food during the sleep period. Mice with two X chromosomes also had accelerated weight gain, fatty liver, and hyperinsulinemia on a high fat diet. The higher expression levels of a subset of genes on the X chromosome that escape inactivation may influence adiposity and metabolic disease. The effect of X chromosome genes is present throughout life, but may become particularly significant with increases in longevity and extension of the period spent with reduced gonadal hormone levels.
doi:10.1371/journal.pgen.1002709
PMCID: PMC3349739  PMID: 22589744
15.  Deficiencies in lamin B1 and lamin B2 cause neurodevelopmental defects and distinct nuclear shape abnormalities in neurons 
Molecular Biology of the Cell  2011;22(23):4683-4693.
Lamin B1 is essential for neuronal migration and progenitor proliferation during the development of the cerebral cortex. The observation of distinct phenotypes of Lmnb1- and Lmnb2-knockout mice and the differences in the nuclear morphology of cortical neurons in vivo suggest that lamin B1 and lamin B2 play distinct functions in the developing brain.
Neuronal migration is essential for the development of the mammalian brain. Here, we document severe defects in neuronal migration and reduced numbers of neurons in lamin B1–deficient mice. Lamin B1 deficiency resulted in striking abnormalities in the nuclear shape of cortical neurons; many neurons contained a solitary nuclear bleb and exhibited an asymmetric distribution of lamin B2. In contrast, lamin B2 deficiency led to increased numbers of neurons with elongated nuclei. We used conditional alleles for Lmnb1 and Lmnb2 to create forebrain-specific knockout mice. The forebrain-specific Lmnb1- and Lmnb2-knockout models had a small forebrain with disorganized layering of neurons and nuclear shape abnormalities, similar to abnormalities identified in the conventional knockout mice. A more severe phenotype, complete atrophy of the cortex, was observed in forebrain-specific Lmnb1/Lmnb2 double-knockout mice. This study demonstrates that both lamin B1 and lamin B2 are essential for brain development, with lamin B1 being required for the integrity of the nuclear lamina, and lamin B2 being important for resistance to nuclear elongation in neurons.
doi:10.1091/mbc.E11-06-0504
PMCID: PMC3226484  PMID: 21976703
16.  NF-E2–Related Factor 2 Promotes Atherosclerosis by Effects on Plasma Lipoproteins and Cholesterol Transport That Overshadow Antioxidant Protection 
Objective
To test the hypothesis that NF-E2–related factor 2 (Nrf2) expression plays an antiatherogenic role by its vascular antioxidant and anti-inflammatory properties.
Methods and Results
Nrf2 is an important transcription factor that regulates the expression of phase 2 detoxifying enzymes and antioxidant genes. Its expression in vascular cells appears to be an important factor in the protection against vascular oxidative stress and inflammation. We developed Nrf2 heterozygous (HET) and homozygous knockout (KO) mice on an apolipoprotein (apo) E–null background by sequential breeding, resulting in Nrf2−/−, apoE−/− (KO), Nrf2−/+, apoE−/− (HET) and Nrf2+/+, and apoE−/− wild-type littermates. KO mice exhibited decreased levels of antioxidant genes with evidence of increased reactive oxygen species generation compared with wild-type controls. Surprisingly, KO males exhibited 47% and 53% reductions in the degree of aortic atherosclerosis compared with HET or wild-type littermates, respectively. Decreased atherosclerosis in KO mice correlated with lower plasma total cholesterol in a sex-dependent manner. KO mice also had a decreased hepatic cholesterol content and a lower expression of lipogenic genes, suggesting that hepatic lipogenesis could be reduced. In addition, KO mice exhibited atherosclerotic plaques characterized by a lesser macrophage component and decreased foam cell formation in an in vitro lipid-loading assay. This was associated with a lower rate of cholesterol influx, mediated in part by decreased expression of the scavenger receptor CD36.
Conclusion
Nrf2 expression unexpectedly promotes atherosclerotic lesion formation in a sex-dependent manner, most likely by a combination of systemic metabolic and local vascular effects.
doi:10.1161/ATVBAHA.110.210906
PMCID: PMC3037185  PMID: 20947826
atherosclerosis; cytokines; lipoproteins; reactive oxygen species; foam cell formation; lipogenesis; Nrf2
17.  Cholesterol Intake Modulates Plasma Triglyceride Levels in GPIHBP1-deficient Mice 
Objective
Adult GPIHBP1-deficient mice (Gpihbp1−/−) have severe hypertriglyceridemia; however, the plasma triglyceride levels are only mildly elevated during the suckling phase when lipoprotein lipase (Lpl) is expressed at high levels in the liver. Lpl expression in the liver can be induced in adult mice with dietary cholesterol. We therefore hypothesized that plasma triglyceride levels in adult Gpihbp1−/− mice would be sensitive to cholesterol intake.
Methods and Results
After 4–8 weeks on a western diet containing 0.15% cholesterol, plasma triglyceride levels in Gpihbp1−/− mice were 10,000–12,000 mg/dl. When 0.005% ezetimibe was added to the diet to block cholesterol absorption, Lpl expression in the liver was reduced significantly, and the plasma triglyceride levels were significantly higher (>15,000 mg/dl). We also assessed plasma triglyceride levels in Gpihbp1−/− mice fed western diets containing either high (1.3%) or low (0.05%) amounts of cholesterol. The high-cholesterol diet significantly increased Lpl expression in the liver and lowered plasma triglyceride levels.
Conclusions
Treatment of Gpihbp1−/− mice with ezetimibe lowers Lpl expression in the liver and increases plasma triglyceride levels. A high-cholesterol diet had the opposite effects. Thus, cholesterol intake modulates plasma triglyceride levels in Gpihbp1−/− mice.
doi:10.1161/ATVBAHA.110.214403
PMCID: PMC2959134  PMID: 20814015
lipoprotein lipase; chylomicronemia; hypertriglyceridemia; GPIHBP1
18.  Agpat6—a Novel Lipid Biosynthetic Gene Required for Triacylglycerol Production in Mammary Epithelium 
Journal of lipid research  2006;47(4):734-744.
In analyzing the sequence tags for mutant mouse embryonic stem (ES) cell lines in BayGenomics (a mouse gene-trapping resource), we identified a novel gene, Agpat6, with sequence similarities to previously characterized glycerolipid acyltransferases. Agpat6’s closest family member is another novel gene that we have provisionally designated Agpat8. Both Agpat6 and Agpat8 are conserved from plants, nematodes, and flies to mammals. AGPAT6, which is predicted to contain multiple membrane-spanning helices, is found exclusively within the endoplasmic reticulum in mammalian cells. To gain insights into the in vivo importance of Agpat6, we used the Agpat6 ES cell line from BayGenomics to create Agpat6-deficient (Agpat6−/−) mice. Agpat6−/− mice lacked full-length Agpat6 transcripts, as judged by northern blots. One of the most striking phenotypes of Agpat6−/− mice was a defect in lactation. Pups nursed by Agpat6−/− mothers die perinatally. Normally, Agpat6 is expressed at high levels in the mammary epithelium of breast tissue, but not in the surrounding adipose tissue. Histological studies revealed that the aveoli and ducts of Agpat6−/− lactating mammary glands were underdeveloped, and there was a dramatic decrease in size and number of lipid droplets within mammary epithelial cells and ducts. Also, the milk from Agpat6−/− mice was markedly depleted in diacylglycerols and triacylglycerols. Thus, we identified a novel glycerolipid acyltransferase of the endoplasmic reticulum, AGPAT6, which is crucial for the production of milk fat by the mammary gland.
doi:10.1194/jlr.M500556-JLR200
PMCID: PMC3196597  PMID: 16449762
LPAAT; acyltransferase; transacylase; milk fat
19.  Paraoxonase 2 Deficiency Alters Mitochondrial Function and Exacerbates the Development of Atherosclerosis 
Antioxidants & Redox Signaling  2011;14(3):341-351.
Abstract
Increased production of reactive oxygen species (ROS) as a result of decreased activities of mitochondrial electron transport chain (ETC) complexes plays a role in the development of many inflammatory diseases, including atherosclerosis. Our previous studies established that paraoxonase 2 (PON2) possesses antiatherogenic properties and is associated with lower ROS levels. The aim of the present study was to determine the mechanism by which PON2 modulates ROS production. In this report, we demonstrate that PON2-def mice on the hyperlipidemic apolipoprotein E−/− background (PON2-def/apolipoprotein E−/−) develop exacerbated atherosclerotic lesions with enhanced mitochondrial oxidative stress. We show that PON2 protein is localized to the inner mitochondrial membrane, where it is found associated with respiratory complex III. Employing surface-plasmon-resonance, we demonstrate that PON2 binds with high affinity to coenzyme Q10, an important component of the ETC. Enhanced mitochondrial oxidative stress in PON2-def mice was accompanied by significantly reduced ETC complex I + III activities, oxygen consumption, and adenosine triphosphate levels in PON2-def mice. In contrast, overexpression of PON2 effectively protected mitochondria from antimycin- or oligomycin-mediated mitochondrial dysfunction. Our results illustrate that the antiatherogenic effects of PON2 are, in part, mediated by the role of PON2 in mitochondrial function. Antioxid. Redox Signal. 14, 341–351.
doi:10.1089/ars.2010.3430
PMCID: PMC3011913  PMID: 20578959
20.  Insulin Resistance and Altered Systemic Glucose Metabolism in Mice Lacking Nur77 
Diabetes  2009;58(12):2788-2796.
OBJECTIVE
Nur77 is an orphan nuclear receptor with pleotropic functions. Previous studies have identified Nur77 as a transcriptional regulator of glucose utilization genes in skeletal muscle and gluconeogenesis in liver. However, the net functional impact of these pathways is unknown. To examine the consequence of Nur77 signaling for glucose metabolism in vivo, we challenged Nur77 null mice with high-fat feeding.
RESEARCH DESIGN AND METHODS
Wild-type and Nur77 null mice were fed a high-fat diet (60% calories from fat) for 3 months. We determined glucose tolerance, tissue-specific insulin sensitivity, oxygen consumption, muscle and liver lipid content, muscle insulin signaling, and expression of glucose and lipid metabolism genes.
RESULTS
Mice with genetic deletion of Nur77 exhibited increased susceptibility to diet-induced obesity and insulin resistance. Hyperinsulinemic-euglycemic clamp studies revealed greater high-fat diet–induced insulin resistance in both skeletal muscle and liver of Nur77 null mice compared with controls. Loss of Nur77 expression in skeletal muscle impaired insulin signaling and markedly reduced GLUT4 protein expression. Muscles lacking Nur77 also exhibited increased triglyceride content and accumulation of multiple even-chained acylcarnitine species. In the liver, Nur77 deletion led to hepatic steatosis and enhanced expression of lipogenic genes, likely reflecting the lipogenic effect of hyperinsulinemia.
CONCLUSIONS
Collectively, these data demonstrate that loss of Nur77 influences systemic glucose metabolism and highlight the physiological contribution of muscle Nur77 to this regulatory pathway.
doi:10.2337/db09-0763
PMCID: PMC2780886  PMID: 19741162
21.  Phosphatidate degradation: Phosphatidate phosphatases (lipins) and lipid phosphate phosphatases 
Biochimica et biophysica acta  2009;1791(9):956-961.
Summary
Three lipid phosphate phosphatases (LPPs) regulate cell signaling by modifying the concentrations of a variety of lipid phosphates versus their dephosphorylated products. In particular, the LPPs are normally considered to regulate signaling by the phospholipase D (PLD) pathway by converting phosphatidate (PA) to diacylglycerol (DAG). LPP activities do modulate the accumulations of PA and DAG following PLD activation, but this could also involve an effect upstream of PLD activation. The active sites of the LPPs are on the exterior surface of plasma membranes, or on the luminal surface of internal membranes. Consequently, the actions of the LPPs in metabolizing PA formed by PLD1 or PLD2 should depend on the access of this substrate to the active site of the LPPs. Alternatively, PA generated on the cytosolic surface of membranes should be readily accessible to the family of specific phosphatidate phosphatases, namely the lipins. Presently, there is only indirect evidence for the lipins participating in cell signaling following PLD activation. So far, we know relatively little about how individual LPPs and specific phosphatidate phosphatases (lipins) modulate cell signaling through controlling the turnover of bioactive lipids that are formed after PLD activation.
doi:10.1016/j.bbalip.2009.02.007
PMCID: PMC2875194  PMID: 19250975
Diacylglycerol; lysophosphatidate; phosphatidate; phospholipase D; triacylglycerol synthesis
22.  Agpat6 deficiency causes subdermal lipodystrophy and resistance to obesityS 
Journal of lipid research  2006;47(4):745-754.
Triglyceride synthesis in most mammalian tissues involves the sequential addition of fatty acids to a glycerol backbone, with unique enzymes required to catalyze each acylation step. Acylation at the sn-2 position requires 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) activity. To date, seven Agpat genes have been identified based on activity and/or sequence similarity, but their physiological functions have not been well established. We have generated a mouse model deficient in AGPAT6, which is normally expressed at high levels in brown adipose tissue (BAT), white adipose tissue (WAT), and liver. Agpat6-deficient mice exhibit a 25% reduction in body weight and resistance to both diet-induced and genetically induced obesity. The reduced body weight is associated with increased energy expenditure, reduced triglyceride accumulation in BAT and WAT, reduced white adipocyte size, and lack of adipose tissue in the subdermal region. In addition, the fatty acid composition of triacylglycerol, diacylglycerol, and phospholipid is altered, with proportionally greater polyunsaturated fatty acids at the expense of monounsaturated fatty acids. Thus, Agpat6 plays a unique role in determining triglyceride content and composition in adipose tissue and liver that cannot be compensated by other members of the Agpat family.
doi:10.1194/jlr.M500553-JLR200
PMCID: PMC2901549  PMID: 16436371
acyltransferase; gene-trap; adipose tissue; energy expenditure; 1-acylglycerol-3-phosphate O-acyltransferase
23.  The Lipin Family: Mutations and Metabolism 
Current opinion in lipidology  2009;20(3):165-170.
Purpose of review
The family of three lipin proteins act as phosphatidate phosphatase (PAP) enzymes required for glycerolipid biosynthesis, and also as transcriptional coactivators that regulate expression of lipid metabolism genes. The genes for lipin-1, lipin-2 and lipin-3 are expressed in key metabolic tissues, including adipose tissue, skeletal muscle, and liver, but the physiological functions of each member of the family have not been fully elucidated. Here we examine the most recent studies that provide information about the roles of lipin proteins in metabolism and human disease.
Recent findings
Recent studies have identified mutations that cause lipin-1 or lipin-2 deficiency in humans, leading to acute myoglobinuria in childhood or the inflammatory disorder Majeed syndrome, respectively. The effects of lipin-1 deficiency appear to include both the loss of glycerolipid building blocks and the accumulation of lipid intermediates that disrupt cellular function. Several studies have demonstrated that polymorphisms in the LPIN1 and LPIN2 genes are associated with metabolic disease traits, including insulin sensitivity, diabetes, blood pressure, and response to thiazolidinedione drugs. Furthermore, lipin-1 expression levels in adipose tissue and/or liver are positively correlated with insulin sensitivity. Studies of lipin-1 in adipocytes have shed some light on its relationship with insulin sensitivity.
Summary
Lipin-1 and lipin-2 are required for normal lipid homeostasis, and have unique physiological roles. Future studies, for example using engineered mouse models, will be required to fully elucidate their specific roles in normal physiology and disease.
doi:10.1097/MOL.0b013e32832adee5
PMCID: PMC2875192  PMID: 19369868
triglyceride; phosphatidic acid phosphatase; transcriptional coactivator; lipodystrophy; obesity; insulin resistance; myopathy
24.  The lipin protein family: dual roles in lipid biosynthesis and gene expression 
FEBS letters  2007;582(1):90-96.
The prevalence of obesity in the western world has focused attention on factors that influence triglyceride biosynthesis, storage, and utilization. Members of the lipin protein family have a newly discovered enzymatic role in triglyceride and phospholipid biosynthesis as a phosphatidate phosphatase, and also act as an inducible transcriptional coactivator in conjunction with PGC-1α and PPARα. Through these activities, the founding member of the family, lipin-1, influences lipid metabolism and glucose homeostasis in diverse tissues including adipose tissue, skeletal muscle, and liver. The physiological roles of lipin-2 and lipin-3 are less well defined, but are likely to carry out similar functions in glycerolipid biosynthesis and gene expression in a distinct tissue distribution.
doi:10.1016/j.febslet.2007.11.014
PMCID: PMC2848953  PMID: 18023282
adipose tissue; ipodystrophy; obesity; triaclyglycerol; phosphatidate phosphatase; transcriptional coactivator
25.  Adipose tissue dysfunction tracks disease progression in two Huntington's disease mouse models 
Human Molecular Genetics  2009;18(6):1006-1016.
In addition to the hallmark neurological manifestations of Huntington's disease (HD), weight loss with metabolic dysfunction is often observed in the later stages of disease progression and is associated with poor prognosis. The mechanism for weight loss in HD is unknown. Using two mouse models of HD, the R6/2 transgenic and CAG140 knock-in mouse strains, we demonstrate that adipose tissue dysfunction is detectable at early ages and becomes more pronounced as the disease progresses. Adipocytes acquire a ‘de-differentiated’ phenotype characterized by impaired expression of fat storage genes. In addition, HD mice exhibit reduced levels of leptin and adiponectin, adipose tissue-derived hormones that regulate food intake and glucose metabolism. Importantly, some of these changes occur prior to weight loss and development of some of the characteristic neurological symptoms. We demonstrate that impaired gene expression and lipid accumulation in adipocytes can be recapitulated by expression of an inducible mutant huntingtin transgene in an adipocyte cell line and that mutant huntingtin inhibits transcriptional activity of the PGC-1α co-activator in adipocytes, which may contribute to aberrant gene expression. Thus, our findings indicate that mutant huntingtin has direct detrimental effects in cell types other than neurons. The results also indicate that circulating adipose-tissue-derived hormones may be accessible markers for HD prognosis and progression and suggest that adipose tissue may be a useful therapeutic target to improve standard of life for HD patients.
doi:10.1093/hmg/ddn428
PMCID: PMC2649017  PMID: 19124532

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