Extensive metabolic remodeling after limiting mitochondrial lipid burden is consistent with an improved metabolic health profile.

Mitochondrial lipid overload in skeletal muscle contributes to insulin resistance, and strategies limiting this lipid pressure improve glucose homeostasis; however, comprehensive cellular adaptations that occur in response to such an intervention have not been reported. Herein, mice with skeletal muscle-specific deletion of carnitine palmitoyltransferase 1b (Cpt1bM-/-), which limits mitochondrial lipid entry, were fed a moderate fat (25%) diet, and samples were subjected to a multimodal analysis merging transcriptomics, proteomics, and nontargeted metabolomics to characterize the coordinated multilevel cellular responses that occur when mitochondrial lipid burden is mitigated. Limiting mitochondrial fat entry predictably improves glucose homeostasis; however, remodeling of glucose metabolism pathways pales compared with adaptations in amino acid and lipid metabolism pathways, shifts in nucleotide metabolites, and biogenesis of mitochondria and peroxisomes. Despite impaired fat utilization, Cpt1bM-/- mice have increased acetyl-CoA (14-fold) and NADH (2-fold), indicating metabolic shifts yield sufficient precursors to meet energy demand; however, this does not translate to enhance energy status as Cpt1bM-/- mice have low ATP and high AMP levels, signifying energy deficit. Comparative analysis of transcriptomic data with disease-associated gene-sets not only predicted reduced risk of glucose metabolism disorders but was also consistent with lower risk for hepatic steatosis, cardiac hypertrophy, and premature death. Collectively, these results suggest induction of metabolic inefficiency under conditions of energy surfeit likely contributes to improvements in metabolic health when mitochondrial lipid burden is mitigated. Moreover, the breadth of disease states to which mechanisms induced by muscle-specific Cpt1b inhibition may mediate health benefits could be more extensive than previously predicted.

Embryonic cell migratory capacity is impaired upon exposure to glucose in vivo and in vitro.

BACKGROUND: Impairments in cell migration during vertebrate gastrulation lead to structural birth defects, such as heart defects and neural tube defects. These defects are more frequent in progeny from diabetic pregnancies, and we have recently provided evidence that maternal diabetes leads to impaired migration of embryonic mesodermal cells in a mouse model of diabetic pregnancy. METHODS: We here report the isolation of primary cell lines from normal and diabetes-exposed embryos of the nonobese diabetic mouse strain, and characterization of their energy metabolism and expression of nutrient transporter genes by quantitative real-time PCR. RESULTS: Expression levels of several genes in the glucose transporter and fatty acid transporter gene families were altered in diabetes-exposed cells. Notably, primary cells from embryos with prior in vivo exposure to maternal diabetes exhibited reduced capacity for cell migration in vitro. CONCLUSIONS: Primary cells isolated from diabetes-exposed embryos retained a "memory" of their in vivo exposure, manifesting in cell migration impairment. Thus, we have successfully established an in vitro experimental model for the mesoderm migration defects observed in diabetes-exposed mouse embryos.

An ethanolic extract of Artemisia scoparia inhibits lipolysis in vivo and has antilipolytic effects on murine adipocytes in vitro.

An ethanolic extract of Artemisia scoparia (SCO) has metabolically favorable effects on adipocyte development and function in vitro and in vivo. In diet-induced obese mice, SCO supplementation significantly reduced fasting glucose and insulin levels. Given the importance of adipocyte lipolysis in metabolic health, we hypothesized that SCO modulates lipolysis in vitro and in vivo. Free fatty acids and glycerol were measured in the sera of mice fed a high-fat diet with or without SCO supplementation. In cultured 3T3-L1 adipocytes, the effects of SCO on lipolysis were assessed by measuring glycerol and free fatty acid release. Microarray analysis, qPCR, and immunoblotting were used to assess gene expression and protein abundance. We found that SCO supplementation of a high-fat diet in mice substantially reduces circulating glycerol and free fatty acid levels, and we observed a cell-autonomous effect of SCO to significantly attenuate tumor necrosis factor-α (TNFα)-induced lipolysis in cultured adipocytes. Although several prolipolytic and antilipolytic genes were identified by microarray analysis of subcutaneous and visceral adipose tissue from SCO-fed mice, regulation of these genes did not consistently correlate with SCO's ability to reduce lipolytic metabolites in sera or cell culture media. However, in the presence of TNFα in cultured adipocytes, SCO induced antilipolytic changes in phosphorylation of hormone-sensitive lipase and perilipin. Together, these data suggest that the antilipolytic effects of SCO on adipose tissue play a role in the ability of this botanical extract to improve whole body metabolic parameters and support its use as a dietary supplement to promote metabolic resiliency.

Lipid peroxidation regulates podocyte migration and cytoskeletal structure through redox sensitive RhoA signaling.

Early podocyte loss is characteristic of chronic kidney diseases (CKD) in obesity and diabetes. Since treatments for hyperglycemia and hypertension do not prevent podocyte loss, there must be additional factors causing podocyte depletion. The role of oxidative stress has been implicated in CKD but it is not known how exactly free radicals affect podocyte physiology. To assess this relationship, we investigated the effects of lipid radicals on podocytes, as lipid peroxidation is a major form of oxidative stress in diabetes. We found that lipid radicals govern changes in podocyte homeostasis through redox sensitive RhoA signaling: lipid radicals inhibit migration and cause loss of F-actin fibers. These effects were prevented by mutating the redox sensitive cysteines of RhoA. We therefore suggest that in diseases associated with increased lipid peroxidation, lipid radicals can determine podocyte function with potentially pathogenic consequences for kidney physiology.

Impaired Mitochondrial Fat Oxidation Induces FGF21 in Muscle.

Fatty acids are the primary fuel source for skeletal muscle during most of our daily activities, and impaired fatty acid oxidation (FAO) is associated with insulin resistance. We have developed a mouse model of impaired FAO by deleting carnitine palmitoyltransferase-1b specifically in skeletal muscle (Cpt1b(m-/-)). Cpt1b(m-/-) mice have increased glucose utilization and are resistant to diet-induced obesity. Here, we show that inhibition of mitochondrial FAO induces FGF21 expression specifically in skeletal muscle. The induction of FGF21 in Cpt1b-deficient muscle is dependent on AMPK and Akt1 signaling but independent of the stress signaling pathways. FGF21 appears to act in a paracrine manner to increase glucose uptake under low insulin conditions, but it does not contribute to the resistance to diet-induced obesity.

Novel Mode of Defective Neural Tube Closure in the Non-Obese Diabetic (NOD) Mouse Strain.

Failure to close the neural tube results in birth defects, with severity ranging from spina bifida to lethal anencephaly. Few genetic risk factors for neural tube defects are known in humans, highlighting the critical role of environmental risk factors, such as maternal diabetes. Yet, it is not well understood how altered maternal metabolism interferes with embryonic development, and with neurulation in particular. We present evidence from two independent mouse models of diabetic pregnancy that identifies impaired migration of nascent mesodermal cells in the primitive streak as the morphogenetic basis underlying the pathogenesis of neural tube defects. We conclude that perturbed gastrulation not only explains the neurulation defects, but also provides a unifying etiology for the broad spectrum of congenital malformations in diabetic pregnancies.

A unique missense allele of BAF155, a core BAF chromatin remodeling complex protein, causes neural tube closure defects in mice.

Failure of embryonic neural tube closure results in the second most common class of birth defects known as neural tube defects (NTDs). While NTDs are likely the result of complex multigenic dysfunction, it is not known whether polymorphisms in epigenetic regulators may be risk factors for NTDs. Here we characterized Baf155(msp3) , a unique ENU-induced allele in mice. Homozygous Baf155(mps3) embryos exhibit highly penetrant exencephaly, allowing us to investigate the roles of an assembled, but malfunctional BAF chromatin remodeling complex in vivo at the time of neural tube closure. Evidence of defects in proliferation and apoptosis were found within the neural tube. RNA-Seq analysis revealed that surprisingly few genes showed altered expression in Baf155 mutant neural tissue, given the broad epigenetic role of the BAF complex, but included genes involved in neural development and cell survival. Moreover, gene expression changes between individual mutants were variable even though the NTD was consistently observed. This suggests that inconsistent gene regulation contributes to failed neural tube closure. These results shed light on the role of the BAF complex in the process of neural tube closure and highlight the importance of studying missense alleles to understand epigenetic regulation during critical phases of development.

Mutation at the folate receptor 4 locus modulates gene expression profiles in the mouse uterus in response to periconceptional folate supplementation.

Periconceptional supplementation of folic acid to the diet of women is considered a great success for a public health intervention. Higher folate status, either by supplementation, or via the mandatory fortification of grain products in the United States, has led to significant reduction in the incidence of neural tube defects. Besides birth defects, folate deficiency has been linked to a variety of morbidities, most notably to increased risk for cancer. However, recent evidence suggests that excess folate may be detrimental - for birth defect incidence or in the progression of cancer. How folate mediates beneficial or detrimental effects is not well understood. It is also unknown what molecular responses are elicited in women taking folate supplements, and thus experience a bolus of folate on top of the status achieved by fortification. To characterize the response to a periconceptional regimen of supplementation with folinic acid, we performed gene expression profiling experiments on uterus tissue of pregnant mice with either wildtype alleles or targeted disruption at the folate receptor 4 locus. We observed that, depending on the genetic background, folinic acid supplementation affects expression of genes that contribute to lipid metabolism, protein synthesis, mitochondrial function, cell cycle, and cell activation. The extent of the response is strongly modulated by the genetic background. Finally, we provide evidence that folinic acid supplementation in the mutant paradigm affects histone methylation status, a potential mechanism of gene regulation in this model.

Genetic and epigenomic footprints of folate.

Dietary micronutrient composition has long been recognized as a determining factor for human health. Historically, biochemical research has successfully unraveled how vitamins serve as essential cofactors for enzymatic reactions in the biochemical machinery of the cell. Folate, also known as vitamin B9, follows this paradigm as well. Folate deficiency is linked to adverse health conditions, and dietary supplementation with folate has proven highly beneficial in the prevention of neural tube defects. With its function in single-carbon metabolism, folate levels affect nucleotide synthesis, with implications for cell proliferation, DNA repair, and genomic stability. Furthermore, by providing the single-carbon moiety in the synthesis pathway for S-adenosylmethionine, the main methyl donor in the cell, folate also impacts methylation reactions. It is this capacity that extends the reach of folate functions into the realm of epigenetics and gene regulation. Methylation reactions play a major role for several modalities of the epigenome. The specific methylation status of histones, noncoding RNAs, transcription factors, or DNA represents a significant determinant for the transcriptional output of a cell. Proper folate status is therefore necessary for a broad range of biological functions that go beyond the biochemistry of folate. In this review, we examine evolutionary, genetic, and epigenomic footprints of folate and the implications for human health.

Maternal diet modulates the risk for neural tube defects in a mouse model of diabetic pregnancy.

Pregnancies complicated by maternal diabetes have long been known to carry a higher risk for congenital malformations, such as neural tube defects. Using the FVB inbred mouse strain and the Streptozotocin-induced diabetes model, we tested whether the incidence of neural tube defects in diabetic pregnancies can be modulated by maternal diet. In a comparison of two commercial mouse diets, which are considered nutritionally replete, we found that maternal consumption of the unfavorable diet was associated with a more than 3-fold higher rate of neural tube defects. Our results demonstrate that maternal diet can act as a modifier of the risk for abnormal development in high-risk pregnancies, and provide support for the possibility that neural tube defects in human diabetic pregnancies might be preventable by optimized maternal nutrition.

Neural tube defect genes and maternal diabetes during pregnancy.

BACKGROUND: Maternal diabetes during pregnancy is a well-known teratogen that increases the risk for birth defects, such as neural tube defects (NTDs). We have previously shown that maternal diabetes profoundly affects gene expression in the developing embryo, in particular a suite of known NTD genes. In rodent experimental systems, NTDs present as phenotypes of incomplete penetrance in diabetic pregnancies. This property is difficult to reconcile with observations of consistently altered gene expression in exposed embryos. We here show that maternal diabetes increases the overall variability of gene expression levels in embryos. RESULTS: Altered gene expression and increased variability of gene expression together may constitute the molecular correlates for incomplete phenotype penetrance. DISCUSSION: Based on this model, we suggest that maternal diabetes reduces the precision of gene regulation in exposed individuals. Loss of precision in embryonic gene regulation may include changes to the epigenome via deregulated expression of chromatin-modifying factors. Unraveling the mechanisms underlying such epigenetic modifications in diabetic pregnancies will help to understand how teratogenic insults compromise embryonic development and possibly provide avenues for therapeutic intervention.

Identification of regulatory elements in the Isl1 gene locus.

Isl1 is a LIM/homeodomain transcription factor with critical roles for the development of the heart, the nervous system and the pancreas. Both deficiency and mis-expression of Isl1 cause profound developmental defects, demonstrating the importance of proper regulation of Isl1 gene expression during development. In order to understand the mechanisms that control Isl1 expression during embryogenesis and in tissue differentiation, we initiated a screen for gene regulatory elements in the Isl1 locus using a novel dual reporter gene vector that allows screens of large genomic regions through reporter gene assays in vitro and in vivo. We identified regions from the Isl1 gene locus that confer transcriptional activity in pancreatic cell lines in vitro. Using transgenic mice, we furthermore discovered an enhancer with in vivo specificity for the developing heart, as well as visceral and posterior mesoderm. Our findings further suggest that Foxo1 as well as Gata4 contribute to the activity of this enhancer in the developing embryo. We conclude that Isl1 gene expression is controlled in modular fashion by several elements with distinct functionality. Embryonic Isl1 expression in several tissues of mesodermal origin is driven by a specific enhancer that is located 3-6kb downstream of the gene.

Regulation of folate receptor 1 gene expression in the visceral endoderm.

BACKGROUND: Nutrient supply to the developing mammalian embryo is a fundamental requirement. Before completion of the chorioallantoic placenta, the visceral endoderm plays a crucial role in nurturing the embryo. We have found that visceral endoderm cells express folate receptor 1, a high-affinity receptor for the essential micronutrient folic acid, suggesting that the visceral endoderm has an important function for folate transport to the embryo. The mechanisms that direct expression of FOLR1 in the visceral endoderm are unknown. METHODS: Sequences were tested for transcriptional activation capabilities in the visceral endoderm utilizing reporter gene assays in a cell model for extraembryonic endoderm in vitro, and in transgenic mice in vivo. RESULTS: With F9 embryo carcinoma cells as a model for extraembryonic endoderm, we demonstrate that the P4 promoter of the human FOLR1 gene is active during differentiation of the cells towards visceral endoderm. However, transgenic mouse experiments show that promoter sequences alone are insufficient to elicit reporter gene transcription in vivo. Using sequence conservation as guide to choose genomic sequences from the human FOLR1 gene locus, we demonstrate that the sequence termed F1CE2 exhibits specific enhancer activity in F9 cells in vitro, in the visceral endoderm, and later the yolk sac in transgenic mouse embryos in vivo. We further show that the transcription factor HNF4-alpha can activate this enhancer sequence. CONCLUSIONS: We have identified a transcriptional enhancer sequence from the FOLR1 locus with specific activity in vitro and in vivo, and suggest that FOLR1 is a target for regulation by HNF4-alpha.

Wnt signaling in caudal dysgenesis and diabetic embryopathy.

BACKGROUND: Congenital defects are a major complication of diabetic pregnancy, and the leading cause of infant death in the first year of life. Caudal dysgenesis, occurring up to 200-fold more frequently in children born to diabetic mothers, is a hallmark of diabetic pregnancy. Given that there is also an at least threefold higher risk for heart defects and NTDs, it is important to identify the underlying molecular mechanisms for aberrant embryonic development. METHODS: We have investigated gene expression in a transgenic mouse model of caudal dysgenesis, and in a pharmacological model using situ hybridization and quantitative real-time PCR. RESULTS: We identified altered expression of several molecules that control developmental processes and embryonic growth. CONCLUSIONS: The results from our models point towards major implication of altered Wnt signaling in the pathogenesis of developmental anomalies associated with embryonic exposure to maternal diabetes.

Folate transport gene inactivation in mice increases sensitivity to colon carcinogenesis.

Low dietary folate intake is associated with an increased risk for colon cancer; however, relevant genetic animal models are lacking. We therefore investigated the effect of targeted ablation of two folate transport genes, folate binding protein 1 (Folbp1) and reduced folate carrier 1 (RFC1), on folate homeostasis to elucidate the molecular mechanisms of folate action on colonocyte cell proliferation, gene expression, and colon carcinogenesis. Targeted deletion of Folbp1 (Folbp1(+/-) and Folbp1(-/-)) significantly reduced (P < 0.05) colonic Folbp1 mRNA, colonic mucosa, and plasma folate concentration. In contrast, subtle changes in folate homeostasis resulted from targeted deletion of RFC1 (RFC1(+/-)). These animals had reduced (P < 0.05) colonic RFC1 mRNA and exhibited a 2-fold reduction in the plasma S-adenosylmethionine/S-adenosylhomocysteine. Folbp1(+/-) and Folbp1(-/-) mice had larger crypts expressed as greater (P < 0.05) numbers of cells per crypt column relative to Folbp1(+/+) mice. Colonic cell proliferation was increased in RFC1(+/-) mice relative to RFC1(+/+) mice. Microarray analysis of colonic mucosa showed distinct changes in gene expression specific to Folbp1 or RFC1 ablation. The effect of folate transporter gene ablation on colon carcinogenesis was evaluated 8 and 38 weeks post-azoxymethane injection in wild-type and heterozygous mice. Relative to RFC1(+/+) mice, RFC1(+/-) mice developed increased (P < 0.05) numbers of aberrant crypt foci at 8 weeks. At 38 weeks, RFC1(+/-) mice developed local inflammatory lesions with or without epithelial dysplasia as well as adenocarcinomas, which were larger relative to RFC1(+/+) mice. In contrast, Folbp1(+/-) mice developed 4-fold (P < 0.05) more lesions relative to Folbp1(+/+) mice. In conclusion, Folbp1 and RFC1 genetically modified mice exhibit distinct changes in colonocyte phenotype and therefore have utility as models to examine the role of folate homeostasis in colon cancer development.

Developmental consequences of in utero sodium arsenate exposure in mice with folate transport deficiencies.

Previous studies have demonstrated that mice lacking a functional folate binding protein 2 gene (Folbp2-/-) were significantly more sensitive to in utero arsenic exposure than were the wild-type mice similarly exposed. When these mice were fed a folate-deficient diet, the embryotoxic effect of arsenate was further exacerbated. Contrary to expectations, studies on 24-h urinary speciation of sodium arsenate did not demonstrate any significant difference in arsenic biotransformation between Folbp2-/- and Folbp2+/+ mice. To better understand the influence of folate pathway genes on arsenic embryotoxicity, the present investigation utilized transgenic mice with disrupted folate binding protein 1 (Folbp1) and reduced folate carrier (RFC) genes. Because complete inactivation of Folbp1 and RFC genes results in embryonic lethality, we used heterozygous animals. Overall, no RFC genotype-related differences in embryonic susceptibility to arsenic exposure were observed. Embryonic lethality and neural tube defect (NTD) frequency in Folbp1 mice was dose-dependent and differed from the RFC mice; however, no genotype-related differences were observed. The RFC heterozygotes tended to have higher plasma levels of S-adenosylhomocysteine (SAH) than did the wild-type controls, although this effect was not robust. It is concluded that genetic modifications at the Folbp1 and RFC loci confers no particular sensitivity to arsenic toxicity compared to wild-type controls, thus disproving the working hypothesis that decreased methylating capacity of the genetically modified mice would put them at increased risk for arsenic-induced reproductive toxicity.

Chlorotoxin-mediated disinhibition of noradrenergic locus coeruleus neurons using a conditional transgenic approach.

The noradrenergic locus coeruleus (LC) has been implicated in the promotion of arousal, in focused attention and learning, and in the regulation of the sleep/waking cycle. The complex biological functions of the central noradrenergic system have been investigated largely through electrophysiological recordings and neurotoxic lesions of LC neurons. Activation of LC neurons through electrical or chemical stimulation has also led to important insights, although these techniques have limited cellular specificity and short-term effects. Here, we describe a novel method aimed at stimulating the central noradrenergic system in a highly selective manner for prolonged periods of time. This was achieved through the conditional expression of a transgene for chlorotoxin (Cltx) in the LC of adult mice. Chlorotoxin is a component of scorpion venom that partially blocks small conductance chloride channels. In this manner, the influence of GABAergic and glycinergic inhibitory inputs on LC cells is greatly reduced, while their ability to respond to excitatory inputs is unaffected. We demonstrate that the unilateral induction of Cltx expression in the LC is associated with a concomitant ipsilateral increase in the expression of markers of noradrenergic activity in LC neurons. Moreover, LC disinhibition is associated with the ipsilateral induction of the immediate early gene NGFI-A in cortical and subcortical target areas. Unlike previous gain of function approaches, transgenic disinhibition of LC cells is highly selective and persists for at least several weeks. This method represents a powerful new tool to assess the long-term effects of LC activation and is potentially applicable to other neuronal systems.