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Centella asiatica (CA) herb is a traditional medicine, long reputed to provide cognitive benefits. We have reported that CA water extract (CAW) treatment improves cognitive function of aged Alzheimer&rsquo, s disease (AD) model Tg2576 and wild-type (WT) mice, and induces an NRF2-regulated antioxidant response in aged WT mice. Here, CAW was administered to AD model 5XFAD female and male mice and WT littermates (age: 7.6 +/ &minus, 0.6 months), and object recall and contextual fear memory were tested after three weeks treatment. CAW&rsquo, s impact on amyloid-&beta, plaque burden, and markers of neuronal oxidative stress and synaptic density, was assessed after five weeks treatment. CAW antioxidant activity was evaluated via nuclear transcription factor (erythroid-derived 2)-like 2 (NRF2) and NRF2-regulated antioxidant response element gene expression. Memory improvement in both genders and genotypes was associated with dose-dependent CAW treatment without affecting plaque burden, and marginally increased synaptic density markers in the hippocampus and prefrontal cortex. CAW treatment increased Nrf2 in hippocampus and other NRF2 targets (heme oxygenase-1, NAD(P)H quinone dehydrogenase 1, glutamate-cysteine ligase catalytic subunit). Reduced plaque-associated SOD1, an indicator of oxidative stress, was observed in the hippocampi and cortices of CAW-treated 5XFAD mice. We postulate that CAW treatment leads to reduced oxidative stress, contributing to improved neuronal health and cognition.

Adipose tissue is a known storage site for vitamin D steroids and is rapidly emerging as a clinically relevant target tissue for 1,25(OH)2D. Links between vitamin D, body weight, and obesity have been proposed based on clinical and population studies; however, mechanistic understanding of the relationship is limited. The vitamin D receptor (VDR) is expressed in adipocyte precursors and is regulated during the differentiation process, but the impact of 1,25(OH)2D on adipogenesis varies considerably with model systems. A lean phenotype associated with enhanced energy expenditure has been confirmed in mice with genetic deletions in VDR, but whether this phenotype reflects actions of the receptor and/or its ligand in adipose tissue remains to be resolved. This chapter will provide a brief update on the role of adipose tissue as a storage depot for vitamin D and will review the effects of 1,25(OH)2D and the VDR on adipogenesis in vitro and adiposity in animal models.

Vitamin D status has been associated with obesity, metabolic syndrome and several cancers including colon and breast. Since adipocytes express VDR and obesity is a known risk factor for cancer, vitamin D actions in adipose tissue may contribute to its cancer protective effects. In the mammary gland, signaling from adipocytes to epithelial cells is necessary for breast cancer initiation, but the impact of vitamin D on this cross-talk is unclear. To examine the role of VDR in adipose tissue, particularly in the context of the mammary gland, we crossed Vdr-flox mice with Fabp4-cre mice to generate mice with adipose-specific Vdr deletion (termed CVF mice). CVF mice and Fabp4-cre control mice (termed CN1 mice) were reared on high calcium "rescue" diets (for comparison to global VDRKO mice) or on high fat diets (to stimulate adiposity). Vdr expression was significantly reduced in adipose tissue of CVF mice compared to CN1 mice. In contrast to global VDRKO mice (which exhibit adipose atrophy), female CVF mice exhibited higher growth rates and increased visceral fat pad weight compared to control mice. Expression of Ucp1 and Pparg were elevated in white adipose tissue of CVF mice supporting these genes as Vdr targets in mature adipocytes. Adipose-specific Vdr deletion did not impair glucose tolerance or alter the weight of brown adipose tissue, liver, pancreas or bone in response to high fat feeding. In contrast to the effect of adipose-specific Vdr deletion on visceral fat pads, the weight of the subcutaneous (mammary) fat pad was not increased in high fat fed CVF female mice compared to control mice. Quantitative analysis of mammary ductal development on whole mounts and H&E stained sections indicated that adipose-deletion of Vdr significantly enhanced mammary epithelial density and branching. Collectively, these data support the hypothesis that Vdr in mature adipocytes alters the metabolic response to high fat diets and exerts anti-proliferative effects on the mammary epithelium.

Mammary gland samples were isolated from wild type, vitamin D receptor knockout (VDRKO) and 1-alphahydroxylase knockout (1-alphaKO) female mice for whole mounts and paraffin embedding (inguinal) and for RNA and protein isolation (thoracic). Time points collected included 6-10wk old nulliparous, 9 and 16 days pregnancy, 5 and 10 days lactation and 3 and 6 days involution. All whole mounts were completed and showed increased branching during pregnancy in the VDRKO glands relative to wild type and 1-alphaKO glands. Paraffin embedded involution samples were stained with hematoxylin and eosin and showed an apparent decrease in alveolar breakdown in the first few days of involution in VDRKO and 1-alphaKO glands compared to wild type controls. Organ culture studies show that treatment of wild type and 1-alphaKO glands with 1,25 dihydroxyvitamin D decreases proliferation and retards tertiary branching. This is not the case in VDRKO glands. Gene expression analysis via microarray and qPCR provides only a glimpse into the complexities of the signaling involved in this process and is being complimented by ongoing protein analysis. These results suggest that 1-alphaKO mammary glands do not develop exclusively similar to VDRKO or wild type glands which verifies our need to complete the remainder of our studies to determine if the VDR is acting to control mammary gland development through the vitamin D pathway or through some other ligand or possibly without a ligand.

These studies focus on identification of vitamin D regulated pathways that impact development or progression of breast cancer. In mouse experiments, we assessed genomic profiles of glandular tissue and established tumors from MMTV-neu mice fed adequate (250 IU/kg) or high (5000 IU/kg) vitamin D (cholecalciferol). Genomic profiles were also obtained in murine mammary cells that differentially express VDR that were cultured in vitro with 100 nM 1,25-dihydroxyvitamin D (1,25D). Ten candidate genes were identified that were commonly regulated in murine cells treated with 1,25D in vitro and in mammary gland of mice fed high dietary vitamin D. In complementary studies, the vitamin D pathway was evaluated in human mammary epithelial cells as a function of transformation. Genes regulated by 1,25D in human mammary epithelial cells included those involved in innate immunity (CD14), differentiation (Bmp6), extracellular matrix remodeling (Plau) and cell survival (Birc3). Transformation reduced VDR content and blunted the induction of some, but not all, target genes by 1,25D in human mammary cells. Collectively, these in vivo and in vitro data demonstrate that vitamin D signaling impacts on common pathways that drive differentiation, alter metabolism, remodel the extracellular matrix and trigger innate immunity in mammary tissue.

Increased adiposity is a feature of aging in both mice and humans, but the molecular mechanisms underlying age-related changes in adipose tissue stores remain unclear. In previous studies, we noted that 18-month-old normocalcemic vitamin D receptor (VDR) knockout (VDRKO) mice exhibited atrophy of the mammary adipose compartment relative to wild-type (WT) littermates, suggesting a role for VDR in adiposity. Here we monitored body fat depots, food intake, metabolic factors, and gene expression in WT and VDRKO mice on the C57BL6 and CD1 genetic backgrounds. Regardless of genetic background, both sc and visceral white adipose tissue depots were smaller in VDRKO mice than WT mice. The lean phenotype of VDRKO mice was associated with reduced serum leptin and compensatory increased food intake. Similar effects on adipose tissue, leptin and food intake were observed in mice lacking Cyp27b1, the 1α-hydroxylase enzyme that generates 1,25-dihydroxyvitamin D3, the VDR ligand. Although VDR ablation did not reduce expression of peroxisome proliferator-activated receptor-γ or fatty acid synthase, PCR array screening identified several differentially expressed genes in white adipose tissue from WT and VDRKO mice. Uncoupling protein-1, which mediates dissociation of cellular respiration from energy production, was greater than 25-fold elevated in VDRKO white adipose tissue. Consistent with elevation in uncoupling protein-1, VDRKO mice were resistant to high-fat diet-induced weight gain. Collectively, these studies identify a novel role for 1,25-dihydroxyvitamin D3 and the VDR in the control of adipocyte metabolism and lipid storage in vivo. Mice lacking the vitamin D receptor or its ligand display reduced adiposity, resistance to diet-induced obesity, and induction of uncoupling protein-1 in white adipose tissue.

Multiple epidemiological studies have demonstrated correlations between vitamin D status and incidence of many types of cancer, including colon and breast. Our long term goal is to understand the mechanisms by which the vitamin D signaling pathway impacts on breast cancer. Upon absorption or endogenous synthesis, vitamin D is metabolized into 25-hydroxyvitamin D (25D) and 1,25-dihydroxyvitamin D (1,25D) in multiple tissues. 1,25D is a high-affinity ligand for the nuclear vitamin D receptor (VDR) that alters gene expression and inhibits growth of normal and transformed mammary cells. However, 25D and other steroid-like compounds such as bile acids, can also activate VDR in vitro. Although studies with VDR knockout (KO) mice have demonstrated that VDR affects proliferation, differentiation and apoptosis in the mammary gland, the specific ligands that trigger VDR signaling in mammary tissue have yet to be identified. Generation of 1,25D from 25D is mediated by the mitochondrial enzyme CYP27B1 (also known as 25-hydroxyvitamin D 1α-hydroxylase) which is present and developmentally regulated in mammary gland. Studies with the VDRKO mouse have formally established that its phenotype is different from mice lacking CYP27B1, supporting the concept that other VDR ligands exist, and/or that the VDR may act in the absence of ligand. In our studies, we are using animal models to determine whether VDR exerts 1,25D-independent effects on growth, apoptosis or differentiation of mammary epithelial cells in vivo. Glands from VDRKO and CYP27B1KO mice during puberty, pregnancy, lactation, involution and aging have been collected for analysis of gross morphology, histology, and genomic profiling in comparison to age-matched control mice. Our data indicate that mammary glands from VDRKO mice display accelerated branching, enhanced sensitivity to estrogen and progesterone and altered gene expression during puberty and early pregnancy. Surprisingly, initial studies indicate that similar changes are not observed in glands from CYP27B1KO mice, suggesting that 25D or other VDR ligands may substitute for 1,25D in control of mammary cell turnover in vivo. Alternatively, our results are consistent with the concept that unoccupied VDR may function in the mammary gland. To further explore the role of VDR and CYP27B1 in the glandular epithelial compartment, mice with mammary epithelial-specific deletion of VDR or CYP27B1 were generated by crossing MMTV-cre mice with mice carrying floxed alleles of VDR or CYP27B1. Quantitation of VDR and CYP27B1 expression in mammary gland, and characterization of the phenotype of these cre-flox mouse strains is in progress. Collectively, these studies have identified novel roles for the VDR and its ligands in mammary gland development that provide insight into the relationship between vitamin D status and breast cancer. Supported by NIH CA69700. Citation Information: Cancer Res 2009;69(23 Suppl):A41.