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Abstract The Four Core Genotypes (FCG) mouse model has become a valuable model to study the mechanistic basis for biological sex differences. This model allows discrimination between influences of gonadal sex (ovaries or testes) from those associated with genetic sex (presence of XX or XY chromosome complement). FCG mice have illuminated distinct effects of gonadal and chromosomal sex on traits ranging from brain structure and behavior to vulnerability to obesity, atherosclerosis, multiple sclerosis, Alzheimer’s and other diseases. A recent study determined that the Y Sry− chromosome used in a specific line of C57BL/6J FCG mice harbors nine genes that have been duplicated from the X chromosome. This report raised concern that scores of publications that previously used the FCG model may therefore be flawed, but did not provide details regarding how studies can be evaluated for potential impact (or lack of impact) of the translocation. Here we (1) provide a practical description of the genetic translocation for researchers using the FCG model, (2) document that a majority of the studies cited in the recent report are unlikely to be affected by the translocation, (3) provide a scheme for interpreting data from studies with FCG mice harboring the Y Sry− translocation, and (4) delineate expression levels of the nine translocated genes across tissue/cell types as a filter for evaluating their potential involvement in specific phenotypes.

Abstract Statin drugs lower blood cholesterol levels for cardiovascular disease prevention. Women are more likely than men to experience adverse statin effects, particularly new-onset diabetes (NOD) and muscle weakness. Here we find that impaired glucose homeostasis and muscle weakness in statin-treated female mice are associated with reduced levels of the omega-3 fatty acid, docosahexaenoic acid (DHA), impaired redox tone, and reduced mitochondrial respiration. Statin adverse effects are prevented in females by administering fish oil as a source of DHA, by reducing dosage of the X chromosome or the Kdm5c gene, which escapes X chromosome inactivation and is normally expressed at higher levels in females than males. As seen in female mice, we find that women experience more severe reductions than men in DHA levels after statin administration, and that DHA levels are inversely correlated with glucose levels. Furthermore, induced pluripotent stem cells from women who developed NOD exhibit impaired mitochondrial function when treated with statin, whereas cells from men do not. These studies identify X chromosome dosage as a genetic risk factor for statin adverse effects and suggest DHA supplementation as a preventive co-therapy.

Early-life conditions such as prenatal nutrition can have long-term effects on metabolic health, and these effects may differ between males and females. Understanding the biological mechanisms underlying sex differences in the response to early-life environment will improve interventions, but few such mechanisms have been identified, and there is no overall framework for understanding sex differences. Biological sex differences may be due to chromosomal sex, gonadal sex, or interactions between the two. This review describes approaches to distinguish between the roles of chromosomal and gonadal sex, and summarizes findings regarding sex differences in metabolism. The Four Core Genotypes (FCG) mouse model allows dissociation of the sex chromosome genotype from gonadal type, whereas the XY* mouse model can be used to distinguish effects of X chromosome dosage vs the presence of the Y chromosome. Gonadectomy can be used to distinguish between organizational (permanent) and activational (reversible) effects of sex hormones. Baseline sex differences in a variety of metabolic traits are influenced by both activational and organizational effects of gonadal hormones, as well as sex chromosome complement. Thus far, these approaches have not been widely applied to examine sex-dependent effects of prenatal conditions, although a number of studies have found activational effects of estradiol to be protective against the development of hypertension following early-life adversity. Genes that escape X chromosome inactivation (XCI), such as Kdm5c, contribute to baseline sex-differences in metabolism, while Ogt, another XCI escapee, leads to sex-dependent responses to prenatal maternal stress. Genome-wide approaches to the study of sex differences include mapping genetic loci influencing metabolic traits in a sex-dependent manner. Seeking enrichment for binding sites of hormone receptors among genes showing sexually-dimorphic expression can elucidate the relative roles of hormones. Using the approaches described herein to identify mechanisms underlying sex-dependent effects of early nutrition on metabolic health may enable the identification of fundamental mechanisms and potential interventions.

Summary: Balance between metabolic and reproductive processes is important for survival, particularly in mammals that gestate their young. How the nervous system coordinates this balance is an active area of study. Herein, we demonstrate that somatostatin (SST) neurons of the tuberal hypothalamus alter feeding in a manner sensitive to metabolic and reproductive states in mice. Whereas chemogenetic activation of SST neurons increased food intake across sexes, ablation decreased food intake only in female mice during proestrus. This ablation effect was only apparent in animals with low body mass. Fat transplantation and bioinformatics analysis of SST neuronal transcriptomes revealed white adipose as a key modulator of these effects. These studies indicate that SST hypothalamic neurons integrate metabolic and reproductive cues by responding to varying levels of circulating estrogens to modulate feeding differentially based on energy stores. Thus, gonadal steroid modulation of neuronal circuits can be context dependent and gated by metabolic status.

Rhee et al. demonstrates that the potent neuroprotective agent CNTF significantly impacts metabolism of degenerating retinas, thus revealing cellular mechanisms underlying enhanced neuronal viability and providing insight for the ongoing CNTF clinical trials.

Abstract Background Biological sex impacts susceptibility and presentation of cardiovascular disease, which remains the leading cause of death for both sexes. To reduce cardiovascular disease risk, statin drugs are commonly prescribed to reduce circulating cholesterol levels through inhibition of cholesterol synthesis. The effectiveness of statin therapy differs between individuals with a sex bias in the frequency of adverse effects. Limited information is available regarding the mechanisms driving sex-specific responses to hypercholesterolemia or statin treatment. Methods Four Core Genotypes mice (XX and XY mice with ovaries and XX and XY mice with testes) on a hypercholesteremic Apoe –/– background were fed a chow diet without or with simvastatin for 8 weeks. Plasma lipid levels were quantified and hepatic differential gene expression was evaluated with RNA-sequencing to identify the independent effects of gonadal and chromosomal sex. Results In a hypercholesterolemic state, gonadal sex influenced the expression levels of more than 3000 genes, and chromosomal sex impacted expression of nearly 1400 genes, which were distributed across all autosomes as well as the sex chromosomes. Gonadal sex uniquely influenced the expression of ER stress response genes, whereas chromosomal and gonadal sex influenced fatty acid metabolism gene expression in hypercholesterolemic mice. Sex-specific effects on gene regulation in response to statin treatment included a compensatory upregulation of cholesterol biosynthetic gene expression in mice with XY chromosome complement, regardless of presence of ovaries or testes. Conclusion Gonadal and chromosomal sex have independent effects on the hepatic transcriptome to influence different cellular pathways in a hypercholesterolemic environment. Furthermore, chromosomal sex in particular impacted the cellular response to statin treatment. An improved understanding of how gonadal and chromosomal sex influence cellular response to disease conditions and in response to drug treatment is critical to optimize disease management for all individuals.

Abstract Grip strength is a valuable preclinical assay to study muscle physiology in disease and aging by directly determining changes in muscle force generation in active laboratory mice. Existing methods to statistically evaluate grip strength, however, have limitations in the power and scope of the physiological features that are assessed. We therefore designed a microcontroller whose serial measure of resistance-based force enables the simultaneous readout of (1) peak grip strength, (2) force profile (the non-linear progress of force exerted throughout a standard grip strength trial), and (3) cumulative force profile (the integral of force with respect to time of a single grip strength trial). We hypothesized that muscle pathologies of different etiologies have distinct effects on these parameters. To test this, we used our apparatus to assess the three muscle parameters in mice with impaired muscle function resulting from surgically induced peripheral pain, genetic peripheral neuropathy, adverse muscle effects induced by statin drug, and metabolic alterations induced by a high-fat diet. Both surgically induced peripheral nerve injury and statin-associated muscle damage diminished grip strength and force profile, without affecting cumulative force profile. Conversely, genetic peripheral neuropathy resulting from lipin 1 deficiency led to a marked reduction to all three parameters. A chronic high-fat diet led to reduced grip strength and force profile when normalized to body weight. In high-fat fed mice that were exerted aerobically and allowed to recover for 30 min, male mice exhibited impaired force profile parameters, which female mice were more resilient. Thus, simultaneous analysis of peak grip strength, force profile and cumulative force profile distinguishes the muscle impairments that result from distinct perturbations and may reflect distinct motor unit recruitment strategies.

In this paper, the authors show that sex differences in mitochondrial DNA levels and function in the heart contribute to sex biases in functions relevant to heart failure, identifying Acsl6 as a mitochondrial sex-biased regulator of diastolic function.

SARS-CoV-2 is an emerging viral pathogen and a major global public health challenge since December of 2019, with limited effective treatments throughout the pandemic. As part of the innate immune response to viral infection, type I interferons (IFN-I) trigger a signaling cascade that culminates in the activation of hundreds of genes, known as interferon stimulated genes (ISGs), that collectively foster an antiviral state. We report here the identification of a group of type I interferon suppressed genes, including fatty acid synthase (FASN), which are involved in lipid metabolism. Overexpression of FASN or the addition of its downstream product, palmitate, increased viral infection while knockout or knockdown of FASN reduced infection. More importantly, pharmacological inhibitors of FASN effectively blocked infections with a broad range of viruses, including SARS-CoV-2 and its variants of concern. Thus, our studies not only suggest that downregulation of metabolic genes may present an antiviral strategy by type I interferon, but they also introduce the potential for FASN inhibitors to have a therapeutic application in combating emerging infectious diseases such as COVID-19.

Abstract Acute kidney injury (AKI) is common in patients, causes systemic sequelae, and predisposes patients to long-term cardiovascular disease. To date, studies of the effects of AKI on cardiovascular outcomes have only been performed in male mice. We recently demonstrated that male mice developed diastolic dysfunction, hypertension and reduced cardiac ATP levels versus sham 1 year after AKI. The effects of female sex on long-term cardiac outcomes after AKI are unknown. Therefore, we examined the 1-year cardiorenal outcomes following a single episode of bilateral renal ischemia–reperfusion injury in female C57BL/6 mice using a model with similar severity of AKI and performed concomitantly to recently published male cohorts. To match the severity of AKI between male and female mice, females received 34 min of ischemia time compared to 25 min in males. Serial renal function, echocardiograms and blood pressure assessments were performed throughout the 1-year study. Renal histology, and cardiac and plasma metabolomics and mitochondrial function in the heart and kidney were evaluated at 1 year. Measured glomerular filtration rates (GFR) were similar between male and female mice throughout the 1-year study period. One year after AKI, female mice had preserved diastolic function, normal blood pressure, and preserved levels of cardiac ATP. Compared to males, females demonstrated pathway enrichment in arginine metabolism and amino acid related energy production in both the heart and plasma, and glutathione in the plasma. Cardiac mitochondrial respiration in Complex I of the electron transport chain demonstrated improved mitochondrial function in females compared to males, regardless of AKI or sham. This is the first study to examine the long-term cardiac effects of AKI on female mice and indicate that there are important sex-related cardiorenal differences. The role of female sex in cardiovascular outcomes after AKI merits further investigation.

Improving muscle function has great potential to improve the quality of life. To identify novel regulators of skeletal muscle metabolism and function, we performed a proteomic analysis of gastrocnemius muscle from 73 genetically distinct inbred mouse strains, and integrated the data with previously acquired genomics and >300 molecular/phenotypic traits via quantitative trait loci mapping and correlation network analysis. These data identified thousands of associations between protein abundance and phenotypes and can be accessed online (https://muscle.coffeeprot.com/) to identify regulators of muscle function. We used this resource to prioritize targets for a functional genomic screen in human bioengineered skeletal muscle. This identified several negative regulators of muscle function including UFC1, an E2 ligase for protein UFMylation. We show UFMylation is up-regulated in a mouse model of amyotrophic lateral sclerosis, a disease that involves muscle atrophy. Furthermore, in vivo knockdown of UFMylation increased contraction force, implicating its role as a negative regulator of skeletal muscle function.

Lipins need to bind cell membranes before they can function as phosphatidic acid phosphatases. Here, the authors elucidate the structural basis of lipin membrane-association and identify a lipin domain with a novel protein fold that is critical for membrane binding and full functionality of lipins.

Abstract To elucidate the contributions of specific lipid species to metabolic traits, we integrated global hepatic lipid data with other omics measures and genetic data from a cohort of about 100 diverse inbred strains of mice fed a high‐fat/high‐sucrose diet for 8 weeks. Association mapping, correlation, structure analyses, and network modeling revealed pathways and genes underlying these interactions. In particular, our studies lead to the identification of Ifi203 and Map2k6 as regulators of hepatic phosphatidylcholine homeostasis and triacylglycerol accumulation, respectively. Our analyses highlight mechanisms for how genetic variation in hepatic lipidome can be linked to physiological and molecular phenotypes, such as microbiota composition.

Zinc metallopeptidase STE24 (ZMPSTE24) is essential for the conversion of farnesyl–prelamin A to mature lamin A, a key component of the nuclear lamina. In the absence of ZMPSTE24, farnesyl–prelamin A accumulates in the nucleus and exerts toxicity, causing a variety of disease phenotypes. By ∼4 months of age, both male and female Zmpste24−/− mice manifest a near-complete loss of adipose tissue, but it has never been clear whether this phenotype is a direct consequence of farnesyl–prelamin A toxicity in adipocytes. To address this question, we generated a conditional knockout Zmpste24 allele and used it to create adipocyte-specific Zmpste24–knockout mice. To boost farnesyl–prelamin A levels, we bred in the “prelamin A–only” Lmna allele. Gene expression, immunoblotting, and immunohistochemistry experiments revealed that adipose tissue in these mice had decreased Zmpste24 expression along with strikingly increased accumulation of prelamin A. In male mice, Zmpste24 deficiency in adipocytes was accompanied by modest changes in adipose stores (an 11% decrease in body weight, a 23% decrease in body fat mass, and significantly smaller gonadal and inguinal white adipose depots). No changes in adipose stores were detected in female mice, likely because prelamin A expression in adipose tissue is lower in female mice. Zmpste24 deficiency in adipocytes did not alter the number of macrophages in adipose tissue, nor did it alter plasma levels of glucose, triglycerides, or fatty acids. We conclude that ZMPSTE24 deficiency in adipocytes, and the accompanying accumulation of farnesyl–prelamin A, reduces adipose tissue stores, but only modestly and only in male mice.

Lipin/Pah phosphatidic acid phosphatases generate diacylglycerol to regulate triglyceride synthesis and cellular signaling. Here authors determine structures of Tetrahymena thermophila Pah2 and identify an N-terminal amphipathic helix essential for membrane association.

Lipin 1 regulates cellular lipid homeostasis through roles in glycerolipid synthesis (through phosphatidic acid phosphatase activity) and transcriptional coactivation. Lipin 1–deficient individuals exhibit episodic disease symptoms that are triggered by metabolic stress, such as stress caused by prolonged fasting. We sought to identify critical lipin 1 activities during fasting. We determined that lipin 1 deficiency induces widespread alternative mRNA splicing in liver during fasting, much of which is normalized by refeeding. The role of lipin 1 in mRNA splicing was largely independent of its enzymatic function. We identified interactions between lipin 1 and spliceosome proteins, as well as a requirement for lipin 1 to maintain homeostatic levels of spliceosome small nuclear RNAs and specific RNA splicing factors. In fasted Lpin1–/– liver, we identified a correspondence between alternative splicing of phospholipid biosynthetic enzymes and dysregulated phospholipid levels; splicing patterns and phospholipid levels were partly normalized by feeding. Thus, lipin 1 influences hepatic lipid metabolism through mRNA splicing, as well as through enzymatic and transcriptional activities, and fasting exacerbates the deleterious effects of lipin 1 deficiency on metabolic homeostasis.

Objective: Lipocalin-2 (LCN2) is a secreted protein involved in innate immunity and has also been associated with several cardiometabolic traits in both mouse and human studies. However, the causal relationship of LCN2 to these traits is unclear, and most studies have examined only males. Methods: Using adeno-associated viral vectors we expressed LCN2 in either adipose or liver in a tissue specific manner on the background of a whole-body Lcn2 knockout or wildtype mice. Metabolic phenotypes including body weight, body composition, plasma and liver lipids, glucose homeostasis, insulin resistance, mitochondrial phenotyping, and metabolic cage studies were monitored. Results: We studied the genetics of LCN2 expression and associated clinical traits in both males and females in a panel of 100 inbred strains of mice (HMDP). The natural variation in Lcn2 expression across the HMDP exhibits high heritability, and genetic mapping suggests that it is regulated in part by Lipin1 gene variation. The correlation analyses revealed striking tissue dependent sex differences in obesity, insulin resistance, hepatic steatosis, and dyslipidemia. To understand the causal relationships, we examined the effects of expression of LCN2 selectively in liver or adipose. On a Lcn2-null background, LCN2 expression in white adipose promoted metabolic disturbances in females but not males. It acted in an autocrine/paracrine manner, resulting in mitochondrial dysfunction and an upregulation of inflammatory and fibrotic genes. On the other hand, on a null background, expression of LCN2 in liver had no discernible impact on the traits examined despite increasing the levels of circulating LCN2 more than adipose LCN2 expression. The mechanisms underlying the sex-specific action of LCN2 are unclear, but our results indicate that adipose LCN2 negatively regulates its receptor, LRP2 (or megalin), and its repressor, ERα, in a female-specific manner and that the effects of LCN2 on metabolic traits are mediated in part by LRP2. Conclusions: Following up on our population-based studies, we demonstrate that LCN2 acts in a highly sex- and tissue-specific manner in mice. Our results have important implications for human studies, emphasizing the importance of sex and the tissue source of LCN2. Keywords: Sex differences, Diet-induced obesity, Insulin resistance, Adipose fibrosis, Adipose inflammation, Mitochondrial dysfunction

Objective: Obesity has increased to pandemic levels and enhanced understanding of adipose regulation is required for new treatment strategies. Although bone morphogenetic proteins (BMPs) influence adipogenesis, the effect of BMP antagonists such as Noggin is largely unknown. The aim of the study was to define the role of Noggin, an extracellular BMP inhibitor, in adipogenesis. Methods: We generated adipose-derived progenitor cells and a mouse model with adipocyte-specific Noggin deletion using the Adiponectin Cre transgenic mouse, and determined the adipose phenotype of Noggin-deficiency. Results: Our studies showed that Noggin is expressed in progenitor cells but declines in adipocytes, possibly allowing for lipid accumulation. Correspondingly, adipocyte-specific Noggin deletion in vivo promoted age-related obesity in both genders with no change in food intake. Although the loss of Noggin caused white adipose tissue hypertrophy, and whitening and impaired function in brown adipose tissue in both genders, there were clear gender differences with the females being most affected. The females had suppressed expression of brown adipose markers and thermogenic genes including peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC1alpha) and uncoupling protein 1 (UCP1) as well as genes associated with adipogenesis and lipid metabolism. The males, on the other hand, had early changes in a few BAT markers and thermogenic genes, but the main changes were in the genes associated with adipogenesis and lipid metabolism. Further characterization revealed that both genders had reductions in VO2, VCO2, and RER, whereas females also had reduced heat production. Noggin was also reduced in diet-induced obesity in inbred mice consistent with the obesity phenotype of the Noggin-deficient mice. Conclusions: BMP signaling regulates female and male adipogenesis through different metabolic pathways. Modulation of adipose tissue metabolism by select BMP antagonists may be a strategy for long-term regulation of age-related weight gain and obesity. : Although, bone morphogenetic proteins (BMPs) influence fat development, the role of their inhibitors is largely unknown. This study shows that reduction of Noggin causes obesity related to age but not food intake, suggesting that select BMP antagonists may regulate age-related weight gain and obesity. Keywords: Noggin, Bone morphogenetic protein, Adipocyte, Adipogenesis, Obesity

Men and women differ in their risk of developing coronary artery disease, in part due to differences in their levels of sex hormones. Here, AlSiraj et al. show that the XX sex genotype regulates lipid metabolism and promotes atherosclerosis independently of sex hormones in mice.

The regulation of cellular lipid storage and membrane lipid composition plays a critical role in metabolic homeostasis, and dysregulation may contribute to disorders such as obesity, fatty liver, type 2 diabetes, and cardiovascular disease. The mammalian lipin proteins (lipin 1, lipin 2, and lipin 3) are phosphatidic acid phosphatase (PAP) enzymes that modulate levels of cellular triacylglycerols and phospholipids, and also regulate lipid intermediates in cellular signaling pathways. Lipin proteins also have the ability to coactivate/corepress transcription. In humans and mice, lipin gene mutations cause severe metabolic phenotypes including rhabdomyolysis (lipin 1), autoinflammatory disease (lipin 2), and impaired intestinal lipoprotein assembly (lipin 2/lipin 3). Characterization of these diseases has revealed roles for lipin PAP activity in fundamental cellular processes such as autophagy, inflammasome activation, and lipoprotein assembly. Lipin protein activity is regulated at pre- and posttranscriptional levels, which suggests a need for their ordered response to specific physiological stimuli. Challenges for the future include better elucidation of the unique biochemical and physiological properties of individual lipin family members and determination of lipin protein structure-function relationships. Further research may propel exploration of lipin proteins as viable therapeutic targets in metabolic or inflammatory disorders.

Summary: Mitochondria play a central role in lipid metabolism and can bind to lipid droplets. However, the role and functional specialization of the population of peridroplet mitochondria (PDMs) remain unclear, as methods to isolate functional PDMs were not developed until recently. Here, we describe an approach to isolate intact PDMs from murine brown adipose tissue based on their adherence to lipid droplets. PDMs isolated using our approach can be used to study their specialized function by respirometry.For complete information on the use and execution of this protocol, please refer to Benador et al. (2018).

Background: Sex differences in obesity and related diseases are well established. Gonadal hormones are a major determinant of these sex differences. However, sex differences in body size and composition are evident prior to exposure to gonadal hormones, providing evidence for gonadal-independent contributions attributable to the XX or XY sex chromosome complement. Large-scale genetic studies have revealed male/female differences in the genetic architecture of adipose tissue amount and anatomical distribution. However, these studies have typically neglected the X and Y chromosomes. Scope of the review: Here we discuss how the sex chromosome complement may influence obesity, lipid levels, and inflammation. Human sex chromosome anomalies such as Klinefelter syndrome (XXY), as well as mouse models with engineered alterations in sex chromosome complement, support an important role for sex chromosomes in obesity and metabolism. In particular, the Four Core Genotypes mouse model—consisting of XX mice with either ovaries or testes, and XY mice with either ovaries or testes—has revealed an effect of X chromosome dosage on adiposity, hyperlipidemia, and inflammation irrespective of male or female gonads. Mechanisms may include enhanced expression of genes that escape X chromosome inactivation. Major conclusions: Although less well studied than effects of gonadal hormones, sex chromosomes exert independent and interactive effects on adiposity, lipid metabolism, and inflammation. In particular, the presence of two X chromosomes has been associated with increased adiposity and dyslipidemia in mouse models and in XXY men. The enhanced expression of genes that escape X chromosome inactivation may contribute, but more work is required. Keywords: Genetics, Gonadal hormones, Adipose tissue, Sex chromosome anomalies, X chromosome inactivation, Mouse models

The acetyltransferase Tip60 mediates signaling pathways by acetylating non-histone proteins. Here the authors show that fatty acids induce Tip60–dependent acetylation of phosphatidic acid phosphatase lipin1 which, then, translocates to the ER and generates diacylglycerols for triglyceride synthesis.

Diet1 modulates intestinal production of the hormone, fibroblast growth factor (FGF)15, which signals in liver to regulate bile acid synthesis. C57BL/6ByJ mice with a spontaneous Diet1-null mutation are resistant to hypercholesterolemia compared with wild-type C57BL/6J mice through enhanced cholesterol conversion to bile acids. To further characterize the role of Diet1 in metabolism, we generated Diet1−/− mice on the C57BL/6J genetic background. C57BL/6J Diet1−/− mice had elevated bile acid levels, reduced Fgf15 expression, and increased gastrointestinal motility and intestinal luminal water content, which are symptoms of bile acid diarrhea (BAD) in humans. Natural genetic variation in Diet1 mRNA expression levels across 76 inbred mouse strains correlated positively with Ffg15 mRNA and negatively with serum bile acid levels. This led us to investigate the role of DIET1 genetic variation in primary BAD patients. We identified a DIET1 coding variant (rs12256835) that had skewed prevalence between BAD cases and controls. This variant causes an H1721Q amino acid substitution that increases the levels of FGF19 protein secreted from cultured cells. We propose that genetic variation in DIET1 may be a determinant of FGF19 secretion levels, and may affect bile acid metabolism in both physiological and pathological conditions.

Immune cells are vital constituents of the adipose microenvironment that influence both local and systemic lipid metabolism. Mice lacking IL10 have enhanced thermogenesis, but the roles of specific cell types in the metabolic response to IL10 remain to be defined. We demonstrate here that selective loss of IL10 receptor α in adipocytes recapitulates the beneficial effects of global IL10 deletion, and that local crosstalk between IL10-producing immune cells and adipocytes is a determinant of thermogenesis and systemic energy balance. Single Nuclei Adipocyte RNA-sequencing (SNAP-seq) of subcutaneous adipose tissue defined a metabolically-active mature adipocyte subtype characterized by robust expression of genes involved in thermogenesis whose transcriptome was selectively responsive to IL10Rα deletion. Furthermore, single-cell transcriptomic analysis of adipose stromal populations identified lymphocytes as a key source of IL10 production in response to thermogenic stimuli. These findings implicate adaptive immune cell-adipocyte communication in the maintenance of adipose subtype identity and function.

Summary: Hair plays important roles, ranging from the conservation of body heat to the preservation of psychological well-being. Hair loss or alopecia affects millions worldwide, but methods that can be used to regrow hair are lacking. We report that quiescent (telogen) hair follicles can be stimulated to initiate anagen and hair growth by small molecules that activate autophagy, including the metabolites α-ketoglutarate (α-KG) and α-ketobutyrate (α-KB), and the prescription drugs rapamycin and metformin, which impinge on mTOR and AMPK signaling. Stimulation of hair growth by these agents is blocked by specific autophagy inhibitors, suggesting a mechanistic link between autophagy and hair regeneration. Consistently, increased autophagy is detected upon anagen entry during the natural hair follicle cycle, and oral α-KB prevents hair loss in aged mice. Our finding that anagen can be pharmacologically activated in telogen skin when natural anagen-inducing signal(s) are absent has implications for the treatment of hair loss patients. : Hair regeneration requires reactivating dormant hair follicle stem cells. Chai et al. discover that pharmacological induction of autophagy is sufficient to activate quiescent telogen hair follicles, initiating new anagen hair growth. Hair loss resulting from shortened anagen, lengthened telogen, and/or impeded anagen induction may thereby be rescued by activating autophagy. Keywords: autophagy, metabolite, hair loss, hair regeneration, α-ketoglutarate, α-ketobutyrate, rapamycin, metformin, mTOR, AMPK

Cholesterol and fatty acid biosynthesis are regulated by the sterol regulatory element-binding proteins (SREBPs), encoded by Srebf1 and Srebf2. We generated mice that were either deficient or hypomorphic for SREBP-2. SREBP-2 deficiency generally caused death during embryonic development. Analyses of Srebf2−/− embryos revealed a requirement for SREBP-2 in limb development and expression of morphogenic genes. We encountered only one viable Srebf2−/− mouse, which displayed alopecia, attenuated growth, and reduced adipose tissue stores. Hypomorphic SREBP-2 mice (expressing low levels of SREBP-2) survived development, but the female mice exhibited reduced body weight and died between 8 and 12 weeks of age. Male hypomorphic mice were viable but had reduced cholesterol stores in the liver and lower expression of SREBP target genes. Reduced SREBP-2 expression affected SREBP-1 isoforms in a tissue-specific manner. In the liver, reduced SREBP-2 expression nearly abolished Srebf1c transcripts and reduced Srebf1a mRNA levels. In contrast, adipose tissue displayed normal expression of SREBP target genes, likely due to a compensatory increase in Srebf1a expression. Our results establish that SREBP-2 is critical for survival and limb patterning during development. Reduced expression of SREBP-2 from the hypomorphic allele leads to early death in females and reduced cholesterol content in the liver, but not in adipose tissue.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The heart switches its energy substrate from glucose to fatty acids at birth, and maternal hyperglycemia is associated with congenital heart disease. However, little is known about how blood glucose impacts heart formation. Using a chemically defined human pluripotent stem-cell-derived cardiomyocyte differentiation system, we found that high glucose inhibits the maturation of cardiomyocytes at genetic, structural, metabolic, electrophysiological, and biomechanical levels by promoting nucleotide biosynthesis through the pentose phosphate pathway. Blood glucose level in embryos is stable in utero during normal pregnancy, but glucose uptake by fetal cardiac tissue is drastically reduced in late gestational stages. In a murine model of diabetic pregnancy, fetal hearts showed cardiomyopathy with increased mitotic activity and decreased maturity. These data suggest that high glucose suppresses cardiac maturation, providing a possible mechanistic basis for congenital heart disease in diabetic pregnancy.

Parkinson's disease (PD) is characterized by the progressive degeneration of nigrostriatal dopaminergic neurons leading to motor deficits. The mechanisms underlying the preferential vulnerability of nigrostriatal dopaminergic neurons in PD remain poorly understood. Recent evidence supports a role for mitochondrial dysfunction and increased oxidative stress in PD pathogenesis. Genetic and pathological studies also point to alpha-synuclein as a critical factor in both familial and sporadic forms of the disease; alpha-synuclein pathology affects mitochondrial function but is widespread in PD brain, raising the question of its role in the greater vulnerability of nigrostriatal neurons in PD. We have examined mitochondrial function and oxidative damage in mice overexpressing human wild type alpha-synuclein broadly throughout the nervous system under the Thy1 promoter (Thy1-aSyn mice) between 4 and 8 months of age. Similar levels of alpha-synuclein accumulation in mitochondria were detected in the ventral midbrain, striatum and cortex of Thy1-aSyn mice. However, analysis of mitochondrial respiration using Seahorse XF analyzer showed defects in mitochondrial respiratory complexes I, II, IV and V specifically in the midbrain, and IV and V in the striatum, of Thy1-aSyn mice compared to wild type littermates; mitochondrial complex I activity assay by ELISA confirmed a 40% inhibition specifically in the ventral midbrain. Mitochondrial dysfunction can contribute to oxidative stress and we observed a 40% increase in 4-hydroxynenal and 2-fold increase in malondialdehyde levels, indicative of a high level of lipid peroxidation, specifically in the ventral midbrain of Thy1-aSyn mice. The levels of peroxiredoxin 2, a neuronal antioxidant enzyme that is involved in removal of H2O2 and other toxic peroxides were decreased in the midbrain whereas its oxidized form increased 4-fold, suggesting that antioxidant defenses were compromised in this region. In contrast, peroxiredoxin 2 increased in the striatum and cortex, which may contribute to their protection in the presence of high levels of alpha-synuclein. Thus, in mice over-expressing alpha-synuclein, mitochondrial dysfunction occurred preferentially in nigrostriatal dopaminergic neurons many months before striatal dopamine loss occurs at 14 months of age. This may contribute to a higher level of oxidative stress that overwhelms antioxidant defense in these neurons, leading to their increased vulnerability in PD.

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.

(1) Background: Paraoxonase 2 (PON2) is a ubiquitously expressed protein localized to endoplasmic reticulum and mitochondria. Previous studies have shown that PON2 exhibits anti-oxidant and anti-inflammatory functions, and PON2-deficient (PON2-def) mice are more susceptible to atherosclerosis. Furthermore, PON2 deficiency leads to impaired mitochondrial function. (2) Methods: In this study, we examined the susceptibility of PON2-def mice to diet-induced obesity. (3) Results: After feeding of an obesifying diet, the PON2-def mice exhibited significantly increased body weight due to increased fat mass weight as compared to the wild-type (WT) mice. The increased adiposity was due, in part, to increased adipocyte hypertrophy. PON2-def mice had increased fasting insulin levels and impaired glucose tolerance after diet-induced obesity. PON2-def mice had decreased oxygen consumption and energy expenditure. Furthermore, the oxygen consumption rate of subcutaneous fat pads from PON2-def mice was lower compared to WT mice. Gene expression analysis of the subcutaneous fat pads revealed decreased expression levels of markers for beige adipocytes in PON2-def mice. (4) Conclusions: We concluded that altered systemic energy balance, perhaps due to decreased beige adipocytes and mitochondrial dysfunction in white adipose tissue of PON2-def mice, leads to increased obesity in these mice.

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.

Interest in lipolysis and the metabolism of triglyceride-rich lipoproteins was recently reignited by the discovery of severe hypertriglyceridemia (chylomicronemia) in glycosylphosphatidylinositol-anchored high density lipoprotein–binding protein 1 (GPIHBP1)-deficient mice. GPIHBP1 is expressed exclusively in capillary endothelial cells and binds lipoprotein lipase (LPL) avidly. These findings prompted speculation that GPIHBP1 serves as a binding site for LPL in the capillary lumen, creating “a platform for lipolysis.” More recent studies have identified a second and more intriguing role for GPIHBP1—picking up LPL in the subendothelial spaces and transporting it across endothelial cells to the capillary lumen. Here, we review the studies that revealed that GPIHBP1 is the LPL transporter and discuss which amino acid sequences are required for GPIHBP1–LPL interactions. We also discuss the human genetics of LPL transport, focusing on cases of chylomicronemia caused by GPIHBP1 mutations that abolish GPIHBP1’s ability to bind LPL, and LPL mutations that prevent LPL binding to GPIHBP1.

Phosphatidate phosphatase-1 (PAP-1) converts phosphatidate to diacylglycerol and plays a key role in the biosynthesis of phospholipids and triacylglycerol (TAG). PAP-1 activity is encoded by members of the lipin family, including lipin-1 (1α and 1β), -2, and -3. We determined the effect of lipin-1 expression on the assembly and secretion of very low density lipoproteins (VLDL) using McA-RH7777 cells. Expression of lipin-1α or -1β increased the synthesis and secretion of [3H]glycerol-labeled lipids under either basal- or oleate-supplemented conditions. In the presence of oleate, the increased TAG secretion was mainly associated with VLDL1 (Sf > 100) and VLDL2 (Sf 20–100). Expression of lipin-1α or -1β increased secretion efficiency and decreased intracellular degradation of [35S]apolipoprotein B-100 (apoB100). Knockdown of lipin-1 using specific short interfering RNA decreased secretion of [3H]glycerolipids and [35S]apoB100 even though total PAP-1 activity was not decreased, owing to the presence of lipin-2 and -3 in the cells. Deletion of the nuclear localization signal sequences within lipin-1α not only abolished nuclear localization but also resulted in impaired association with microsomal membranes. Cells expressing the cytosolic lipin-1α mutant failed to promote [35S]apoB100 synthesis or secretion, and showed compromised stimulation in [3H]TAG synthesis and secretion. Thus, alteration in hepatic expression of lipin-1 and its compartmentalization control VLDL assembly/secretion.

Phosphatidate phosphatase-1 (PAP1) enzymes have a key role in glycerolipid synthesis through the conversion of phosphatidate to diacylglycerol, the immediate precursor of triacylglycerol, phosphatidylcholine, and phosphatidylethanolamine. PAP1 activity in mammals is determined by the lipin family of proteins, lipin-1, lipin-2, and lipin-3, which each have distinct tissue expression patterns and appear to have unique physiological functions. In addition to its role in glycerolipid synthesis, lipin-1 also operates as a transcriptional coactivator, working in collaboration with known nuclear receptors and coactivators to modulate lipid metabolism gene expression. The requirement for different lipin activities in vivo is highlighted by the occurrence of lipodystrophy, insulin resistance, and neuropathy in a lipin-1-deficient mutant mouse strain. In humans, variations in lipin-1 expression levels and gene polymorphisms are associated with insulin sensitivity, metabolic rate, hypertension, and risk for the metabolic syndrome. Furthermore, critical mutations in lipin-2 result in the development of an inflammatory disorder in human patients. A key goal of future studies will be to further elucidate the specific roles and modes of regulation of each of the three lipin proteins in key metabolic processes, including triglyceride and phospholipid synthesis, fatty acid metabolism, and insulin sensitivity.

Lipin-1 deficiency in the mouse causes generalized lipodystrophy, characterized by impaired adipose tissue development and insulin resistance. Lipin-1 expression in differentiating preadipocytes is required for normal expression of adipogenic transcription factors, including peroxisome proliferator-activated receptor γ and CCAAT enhancer binding protein α, and for the synthesis of triacylglycerol. The requirement of lipin-1 for adipocyte differentiation can be explained, in part, by its activity as the sole adipocyte phosphatidic acid phosphatase-1 enzyme, which converts phosphatidate to diacylglycerol, the immediate precursor of triacylglycerol. Here we identify glucocorticoids as the stimulus for the induction of lipin-1 expression in differentiating adipocytes, and characterize a glucocorticoid response element (GRE) in the Lpin1 promoter. The Lpin1 GRE binds to the glucocorticoid receptor and leads to transcriptional activation in adipocytes and hepatocytes, as demonstrated by reporter gene transcription, electrophoretic mobility shift, and chromatin immunoprecipitation assays. This represents the first gene regulatory element identified to directly influence lipin-1 expression levels, and may modulate lipin-1 mRNA levels in adipose tissue and liver in conditions associated with increased local glucocorticoid concentrations in vivo, such as obesity and fasting.

Glucocorticoids (GCs) increase hepatic phosphatidate phosphatase (PAP1) activity. This is important in enhancing the liver's capacity for storing fatty acids as triacylglycerols (TAGs) that can be used subsequently for β-oxidation or VLDL secretion. PAP1 catalyzes the conversion of phosphatidate to diacylglycerol, a key substrate for TAG and phospholipid biosynthesis. PAP1 enzymes in liver include lipin-1A and -1B (alternatively spliced isoforms) and two distinct gene products, lipin-2 and lipin-3. We determined the mechanisms by which the composite PAP1 activity is regulated using rat and mouse hepatocytes. Levels of lipin-1A and -1B mRNA were increased by dexamethasone (dex; a synthetic GC), and this resulted in increased lipin-1 synthesis, protein levels, and PAP1 activity. The stimulatory effect of dex on lipin-1 expression was enhanced by glucagon or cAMP and antagonized by insulin. Lipin-2 and lipin-3 mRNA were not increased by dex/cAMP, indicating that increased PAP1 activity is attributable specifically to enhanced lipin-1 expression. This work provides the first evidence for the differential regulation of lipin activities. Selective lipin-1 expression explains the GC and cAMP effects on increased hepatic PAP1 activity, which occurs in hepatic steatosis during starvation, diabetes, stress, and ethanol consumption.

Elucidation of the metabolic pathways of triacylglycerol (TAG) synthesis is critical to the understanding of chronic metabolic disorders such as obesity, cardiovascular disease, and diabetes. sn-Glycerol-3-phosphate acyltransferase (GPAT) and sn-1-acylglycerol-3-phosphate acyltransferase (AGPAT) catalyze the first and second steps in de novo TAG synthesis. AGPAT6 is one of eight AGPAT isoforms identified through sequence homology, but the enzyme activity for AGPAT6 has not been confirmed. We found that in liver and brown adipose tissue from Agpat6-deficient (Agpat6−/−) mice, N-ethylmaleimide (NEM)-sensitive GPAT specific activity was 65% lower than in tissues from wild-type mice, but AGPAT specific activity was similar. Overexpression of Agpat6 in Cos-7 cells increased an NEM-sensitive GPAT specific activity, but AGPAT specific activity was not increased. Agpat6 and Gpat1 overexpression in Cos-7 cells increased the incorporation of [14C]oleate into diacylglycerol (DAG) or into DAG and TAG, respectively, suggesting that the lysophosphatidic acid, phosphatidic acid, and DAG intermediates initiated by each of these isoforms lie in different cellular pools. Together, these data show that “Agpat6−/− mice” are actually deficient in a novel NEM-sensitive GPAT, GPAT4, and indicate that the alterations in lipid metabolism in adipose tissue, liver, and mammary epithelium of these mice are attributable to the absence of GPAT4.

Brown adipose tissue (BAT) activation via cold exposure is increasingly scrutinized as a potential approach to ameliorate cardio-metabolic risk. Transition to cold temperatures requires changes in the partitioning of energy substrates, re-routing fatty acids to BAT to fuel non-shivering thermogenesis. However, the mechanisms behind the redistribution of energy substrates to BAT remain largely unknown. Angiopoietin-like 4 (ANGPTL4), a protein that inhibits lipoprotein lipase (LPL) activity, is highly expressed in BAT. Here, we demonstrate that ANGPTL4 is part of a shuttling mechanism that directs fatty acids derived from circulating triglyceride-rich lipoproteins to BAT during cold. Specifically, we show that cold markedly down-regulates ANGPTL4 in BAT, likely via activation of AMPK, enhancing LPL activity and uptake of plasma triglyceride-derived fatty acids. In contrast, cold up-regulates ANGPTL4 in WAT, abolishing a cold-induced increase in LPL activity. Together, our data indicate that ANGPTL4 is an important regulator of plasma lipid partitioning during sustained cold.

The availability of genomic resources has already had a tremendous impact on biomedical research. In this review, we describe how whole genome sequence and high-throughput functional genomics projects have facilitated the identification and characterization of important genes in lipid metabolism and disease. We review key approaches and lipid genes identified in the first years of this century and discuss how genomic resources are likely to streamline gene identification and functional characterization in the future.

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.

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, 1-acylglycerol-3-phosphate O-acyltransferase (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 (ER) 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 the 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 ER, AGPAT6, which is crucial for the production of milk fat by the mammary gland.

A subclass of hydroxysteroid dehydrogenases (HSD) are NADP(H)-dependent oxidoreductases that belong to the aldo-keto reductase (AKR) superfamily. They are involved in prereceptor or intracrine steroid modulation, and also act as bile acid-binding proteins. The HSD family members characterized thus far in human and rat have a high degree of protein sequence similarity but exhibit distinct substrate specificity. Here we report the identification of nine murine AKR genes in a cluster on chromosome 13 by a combination of molecular cloning and in silico analysis of this region. These include four previously isolated mouse HSD genes (Akr1c18, Akr1c6, Akr1c12, Akr1c13), the more distantly related Akr1e1, and four novel HSD genes. These genes exhibit highly conserved exon/intron organization and protein sequence predictions indicate 75% amino acid similarity. The previously identified AKR protein active site residues are invariant among all nine proteins, but differences are observed in regions that have been implicated in determining substrate specificity. Differences also occur in tissue expression patterns, with expression of some genes restricted to specific tissues and others expressed at high levels in multiple tissues.Our findings dramatically expand the repertoire of AKR genes and identify unrecognized family members with potential roles in the regulation of steroid metabolism.

The fatty liver dystrophy (fld) mutant mouse is characterized by neonatal fatty liver and hypertriglyceridemia that resolve at weaning, and neuropathy affecting peripheral nerve in adulthood. We now report additional significant manifestations of this single gene mutation, which include adipose tissue deficiency, glucose intolerance, and increased susceptibility to atherosclerosis. In adult fld/fld mice, both white and brown fat pads exhibit an 80% reduction in mass compared with wild-type controls, and consist of immature adipocytes as assessed by morphological and molecular criteria. The lack of lipid accumulation in fld/fld adipose tissue could be attributed, in part, to a failure to induce expression of lipoprotein lipase and enzymes involved in fatty acid synthesis, such as fatty acid synthase and acetyl-CoA carboxylase. Related to the deficiency of adipose tissue, fld/fld mice were also found to exhibit profound glucose intolerance, modest hyperinsulinemia, and reduced tissue response to insulin. As insulin resistance is a important risk factor in vascular disease, we examined susceptibility of fld/fld mice to diet-induced atherosclerosis.Mutant mice fed an atherogenic diet developed 2-fold greater aortic lesions than their wild-type counterparts, despite having a less atherogenic lipoprotein cholesterol profile. The fld adipose-deficient phenotype has both similarities to and distinctions from the group of rare human diseases known as lipodystrophies.

Dietary cholesterol is known to raise total and low density lipoprotein cholesterol concentrations in humans and experimental animals, but the response among individuals varies greatly. Here we describe a mouse strain, C57BL/6ByJ (B6By), that is resistant to diet-induced hypercholesterolemia, in contrast to the phenotype seen in other common strains of mice including the closely related C57BL/6J (B6J) strain. Compared to B6J, B6By mice exhibit somewhat lower basal cholesterol levels on a chow diet, and show a relatively modest increase in absolute levels of total and LDL/VLDL cholesterol in response to an atherogenic diet containing 15% fat, 1.25% cholesterol, and 0.5% cholate. Correspondingly, B6By mice are also resistant to diet-induced aortic lesions, with less than 15% as many lesions as B6J. Food intake and cholesterol absorption are similar between B6By and B6J mice. To investigate the gene(s) underlying the resistant B6By phenotype, we performed genetic crosses with the unrelated mouse strain, A/J. A genome-wide scan revealed a locus, designated Diet1, on chromosome 2 near marker D2Mit117 showing highly significant linkage (lod = 9.6) between B6By alleles and hyporesponse to diet. Examination of known genes in this region suggested that this locus represents a novel gene affecting plasma lipids and atherogenesis in response to diet.—Mouzeyan, A., J. Choi, H. Allayee, X. Wang, J. Sinsheimer, J. Phan, L. W. Castellani, K. Reue, A. J. Lusis, and R. C. Davis. A locus conferring resistance to diet-induced hypercholesterolemia and atherosclerosis on mouse chromosome 2. J. Lipid Res. 2000. 41: 573–582.

Foam cells formed from receptor-mediated uptake of lipoprotein cholesterol by macrophages in the arterial intima are critical in the initiation, progression, and stability of atherosclerotic lesions. Macrophages accumulate cholesterol when conditions favor esterification by acyl-CoA:cholesterol acyltransferase (ACAT) over cholesterylester hydrolysis by a neutral cholesteryl-ester hydrolase, such as hormone-sensitive lipase (HSL), and subsequent cholesterol efflux mediated by extracellular acceptors. We recently made stable transfectants of a murine macrophage cell line, RAW 264.7, that overexpressed a rat HSL cDNA and had a 5-fold higher rate of cholesteryl-ester hydrolysis than control cells. The current study examined the effect of macrophage-specific HSL overexpression on susceptibility to diet-induced atherosclerosis in mice. A transgenic line overexpressing the rat HSL cDNA regulated with a macrophage-specific scavenger receptor promoter-enhancer was established by breeding with C57BL/6J mice. Transgenic peritoneal macrophages exhibited macrophage-specific 7-fold overexpression of HSL cholesterol esterase activity. Total plasma cholesterol levels in transgenic mice fed a chow diet were modestly elevated 16% compared to control littermates. After 14 weeks on a high-fat, high-cholesterol diet, total cholesterol increased 3-fold, with no difference between transgenics and controls. However, HSL overexpression resulted in thicker aortic fatty lesions that were 2.5-times larger in transgenic mice. HSL expression in the aortic lesions was shown by immunocytochemistry. Atherosclerosis was more advanced in transgenic mice exhibiting raised lesions involving the aortic wall, along with lipid accumulation in coronary arteries occurring only in transgenics. Thus, increasing cholesteryl-ester hydrolysis, without concomitantly decreasing ACAT activity or increasing cholesterol efflux, is not sufficient to protect against atherosclerosis.—Escary, J-L., H. A. Choy, K. Reue, X-P. Wang, L. W. Castellani, C. K. Glass, A. J. Lusis, and M. C. Schotz. Paradoxical effect on atherosclerosis of hormone-sensitive lipase overexpression in macrophages. J. Lipid Res. 1999. 40: 397–404.

Fatty liver dystrophy (fld) is an autosomal recessive mutation in mice characterized by hypertriglyceridemia and fatty liver during neonatal development. The fatty liver in fld/fld mice spontaneously resolves between the age of 14–18 days, at which point the animals develop a neuropathy associated with abnormal myelin formation in peripheral nerve. We have investigated the morphological and biochemical alterations that occur in the fatty liver of neonatal fld/fld mice. Studies at the light and electron microscopic level demonstrated the accumulation of lipid droplets and hypertrophic parenchymal cells in fld neonates, with no apparent liver pathology after resolution of the fatty liver. To better characterize the biochemical basis for the development of fatty liver in fld mice, we compared protein expression patterns in the fatty liver of fld mice and in the liver of phenotypically normal (wild-type) littermates using quantitative two-dimensional gel electrophoresis. We detected 24 proteins with significantly altered expression levels (P < 0.001) in the fld fatty liver, 15 of which are proteins that are altered in abundance by peroxisome proliferating chemicals. As these compounds characteristically elicit changes in the expression of mitochondrial and peroxisomal enzymes involved in fatty acid oxidation, we quantitated rates of fatty acid oxidation in hepatocytes isolated from fld and wild-type mice. These studies revealed that hepatic fatty acid oxidation in fld neonates is reduced by 60“% compared to wild-type littermates. In hepatocytes from adult fld mice that no longer exhibit a fatty liver, oxidation rates were similar to those in hepatocytes from age-matched wild-type mice. These findings indicate that altered expression of proteins involved in fatty acid oxidation is associated with triglyceride accumulation in the fld fatty liver. —Rehnmark, S., C. S. Giometti, B. G. Slavin, M. H. Doolittle, and K. Reue. The fatty liver dystrophy mutant mouse: microvesicular steatosis associated with altered expression levels of peroxisome proliferator-regulated proteins. J. Lipid Res. 1998. 39: 2209–2217.