Xianglan Liu

Environmental and Genetic Activation of a Brain-Adipocyte BDNF/Leptin Axis Causes Cancer Remission and Inhibition

Cancer is influenced by its microenvironment, yet broader, environmental effects also play a role but remain poorly defined. We report here that mice living in an enriched housing environment show reduced tumor growth and increased remission. We found this effect in melanoma and colon cancer models, and that it was not caused by physical activity alone. Serum from animals held in an enriched environment (EE) inhibited cancer proliferation in vitro and was markedly lower in leptin. Hypothalamic brain-derived neurotrophic factor (BDNF) was selectively upregulated by EE, and its genetic overexpression reduced tumor burden, whereas BDNF knockdown blocked the effect of EE. Mechanistically, we show that hypothalamic BDNF downregulated leptin production in adipocytes via sympathoneural beta-adrenergic signaling. These results suggest that genetic or environmental activation of this BDNF/leptin axis may have therapeutic significance for cancer.

Molecular therapy of obesity and diabetes by a physiological autoregulatory approach

Hypothalamic brain-derived neurotrophic factor (BDNF) is a key element in the regulation of energy balance. Here we investigated the therapeutic efficacy of BDNF by gene transfer in mouse models of obesity and diabetes. Gene transfer of BDNF led to marked weight loss and alleviation of obesity-associated insulin resistance. To facilitate clinical translation and ensure that BDNF protein expression was appropriately decreased as weight loss progressed, thus preventing cachexia, we developed a molecular autoregulatory system involving a single recombinant adeno-associated virus vector harboring two expression cassettes, one constitutively driving BDNF and the other driving a specific microRNA targeting BDNF. The microRNA element was controlled by a promoter (that controlling the Agrp gene encoding agouti-related peptide) responsive to BDNF-induced physiological changes. Hence, as body weight decreased and agouti-related protein is induced, microRNA expression was activated, inhibiting transgene expression. In contrast to the progressive weight loss associated with a nonregulated approach, this microRNA-approach led to a sustainable plateau of body weight after notable weight loss was achieved. This strategy mimics the bodys endogenous physiological feedback mechanisms, thereby resetting the hypothalamic set point to reverse obesity and metabolic syndrome.

Environmental enrichment exerts sex-specific effects on emotionality in C57BL/6J mice

Environmental enrichment (EE) has been shown to exert various behavioral and mood effects in rodents including emotionality, which has a high propensity to be influenced by sex. However, there are only a few comparative studies evaluating the effect of EE and their results are both inconsistent and inconclusive. In the present study, male and female C57BL/6J adolescent mice were housed in either physical enrichment or standard conditions for four weeks with analysis of affective behaviors in the open field, elevated T-maze and forced swim tests. Hippocampal gene expression was characterized in an additional group of mice. In the open field test, exploration was similarly inhibited by EE in male and female mice. Both sex and housing condition influenced the time mice spent in the center of the arena. In the elevated T-maze, anxiety-like behavior was increased in female and decreased in male mice following EE. We observed a trend for EE-induced inhibition of glucocorticoid receptor (GR) mRNA expression in male but not in female mice. In contrast, mineralocorticoid receptor (MR) expression was unaffected by 10 days of physical enrichment but was lower in female mice compared to male mice. Our data suggest that the balance between hippocampal GR and MR may contribute to the observed sex-specific effect of physical enrichment on emotionality-related behavior.

Adipose VEGF Links the White-to-Brown Fat Switch With Environmental, Genetic, and Pharmacological Stimuli in Male Mice

Living in an enriched environment (EE) decreases adiposity, increases energy expenditure, causes resistance to diet induced obesity, and induces brown-like (beige) cells in white fat via activating a hypothalamic-adipocyte axis. Here we report that EE stimulated vascular endothelial growth factor (VEGF) expression in a fat depot-specific manner prior to the emergence of beige cells. The VEGF up-regulation was independent of hypoxia but required intact sympathetic tone to the adipose tissue. Targeted adipose overexpression of VEGF reproduced the browning effect of EE. Adipose-specific VEGF knockout or pharmacological VEGF blockade with antibodies abolished the induction of beige cell by EE. Hypothalamic brain-derived neurotrophic factor stimulated by EE regulated the adipose VEGF expression, and VEGF signaling was essential to the hypothalamic brain-derived neurotrophic factor-induced white adipose tissue browning. Furthermore, VEGF signaling was essential to the beige cells induction by exercise, a β3-adrenergic agonist, and a peroxisome proliferator-activated receptor-γ ligand, suggesting a common downstream pathway integrating diverse upstream mechanisms. Exploiting this pathway may offer potential therapeutic interventions to obesity and metabolic diseases.

Adipose tissue insulin receptor knockdown via a new primate-derived hybrid recombinant AAV serotype

Adipose tissue plays an essential role in metabolic homeostasis and holds promise as an alternative depot organ in gene therapy. However, efficient methods of gene transfer into adipose tissue in vivo have yet to be established. Here, we assessed the transduction efficiency to fat depots by a family of novel engineered hybrid capsid serotypes (Rec1~4) recombinant adeno-associated viral (AAV) vectors in comparison with natural serotypes AAV1, AAV8, and AAV9. Rec2 serotype led to widespread transduction in both brown fat and white fat with the highest efficiency among the seven serotypes tested. As a proof-of-efficacy, Rec2 serotype was used to deliver Cre recombinase to adipose tissues of insulin receptor floxed animals. Insulin receptor knockdown led to decreased fat pad mass and morphological and molecular changes in the targeted depot. These novel hybrid AAV vectors can serve as powerful tools to genetically manipulate adipose tissue and provide valuable vehicles to gene therapy targeting adipose tissue.

Hypothalamic gene transfer of BDNF inhibits breast cancer progression and metastasis in middle age obese mice.

Activation of the hypothalamus-adipocyte axis is associated with an antiobesity and anticancer phenotype in animal models of melanoma and colon cancer. Brain-derived neurotrophic factor (BDNF) is a key mediator in the hypothalamus leading to preferential sympathoneural activation of adipose tissue and the ensuing resistance to obesity and cancer. Here, we generated middle age obese mice by high fat diet feeding for a year and investigated the effects of hypothalamic gene transfer of BDNF on a hormone receptor-positive mammary tumor model. The recombinant adeno-associated viral vector-mediated overexpression of BDNF led to marked weight loss and decrease of adiposity without change of food intake. BDNF gene therapy improved glucose tolerance, alleviated steatosis, reduced leptin level, inhibited mouse breast cancer EO771 growth, and prevented the metastasis. The reduced tumor growth in BDNF-treated mice was associated with reduced angiogenesis, decreased proliferation, increased apoptosis, and reduced adipocyte recruitment and lipid accumulation. Moreover, BDNF gene therapy reduced inflammation markers in the hypothalamus, the mammary gland, the subcutaneous fat, and the mammary tumor. Our results suggest that manipulating a single gene in the brain may influence multiple mechanisms implicated in obesity-cancer association and provide a target for the prevention and treatment of both obesity and cancer.

Role of Hypothalamic VGF in Energy Balance and Metabolic Adaption to Environmental Enrichment in Mice

Environmental enrichment (EE), a housing condition providing complex physical, social, and cognitive stimulation, leads to improved metabolic health and resistance to diet-induced obesity and cancer. One underlying mechanism is the activation of the hypothalamic-sympathoneural-adipocyte axis with hypothalamic brain-derived neurotrophic factor (BDNF) as the key mediator. VGF, a peptide precursor particularly abundant in the hypothalamus, was up-regulated by EE. Overexpressing BDNF or acute injection of BDNF protein to the hypothalamus up-regulated VGF, whereas suppressing BDNF signaling down-regulated VGF expression. Moreover, hypothalamic VGF expression was regulated by leptin, melanocortin receptor agonist, and food deprivation mostly paralleled to BDNF expression. Recombinant adeno-associated virus-mediated gene transfer of Cre recombinase to floxed VGF mice specifically decreased VGF expression in the hypothalamus. In contrast to the lean and hypermetabolic phenotype of homozygous germline VGF knockout mice, specific knockdown of hypothalamic VGF in male adult mice led to increased adiposity, decreased core body temperature, reduced energy expenditure, and impaired glucose tolerance, as well as disturbance of molecular features of brown and white adipose tissues without effects on food intake. However, VGF knockdown failed to block the EE-induced BDNF up-regulation or decrease of adiposity indicating a minor role of VGF in the hypothalamic-sympathoneural-adipocyte axis. Taken together, our results suggest hypothalamic VGF responds to environmental demands and plays an important role in energy balance and glycemic control likely acting in the melanocortin pathway downstream of BDNF.

Environmental and Genetic Activation of Hypothalamic BDNF Modulates T-cell Immunity to Exert an Anticancer Phenotype

Housing mice in enriched environments with multiple stimuli modulated T-cell immunity and inhibited cancer progression. Enhanced immunity was mediated by hypothalamic BDNF, supporting the concept that manipulating a single gene in the brain can improve cancer immunotherapy. Macroenvironmental factors, including a patients physical and social environment, play a role in cancer risk and progression. Our previous studies show that living in an enriched environment (EE) providing complex stimuli confers an anticancer phenotype in mice mediated, in part by a specific neuroendocrine axis, with brain-derived neurotrophic factor (BDNF) as the key brain mediator. Here, we investigated how an EE modulated T-cell immunity and its role in the EE-induced anticancer effects. Our data demonstrated that CD8 T cells were required to mediate the anticancer effects of an EE in an orthotropic model of melanoma. In secondary lymphoid tissue (SLT), an EE induced early changes in the phenotype of T-cell populations, characterized by a decrease in the ratio of CD4 T helper to CD8 cytotoxic T lymphocytes (CTL). Overexpression of hypothalamic BDNF reproduced EE-induced T-cell phenotypes in SLT, whereas knockdown of hypothalamic BDNF inhibited EE-induced immune modulation in SLT. Both propranolol and mifepristone blocked the EE-associated modulation of CTLs in SLT, suggesting that both the sympathetic nervous system and the hypothalamic–pituitary–adrenal axis were involved. Our results demonstrated that enhanced anticancer effect of an EE was mediated at least in part through modulation of T-cell immunity and provided support to the emerging concept of manipulating a single gene in the brain to improve cancer immunotherapy. Cancer Immunol Res; 4(6); 488–97. ©2016 AACR.

Hepatic Expression of Adenovirus 36 E4ORF1 Improves Glycemic Control and Promotes Glucose Metabolism via AKT Activation.

Considering that impaired proximal insulin signaling is linked with diabetes, approaches that enhance glucose disposal independent of insulin signaling are attractive. In vitro data indicate that the E4ORF1 peptide derived from human adenovirus 36 (Ad36) interacts with cells from adipose tissue, skeletal muscle, and liver to enhance glucose disposal, independent of proximal insulin signaling. Adipocyte-specific expression of Ad36E4ORF1 improves hyperglycemia in mice. To determine the hepatic interaction of Ad36E4ORF1 in enhancing glycemic control, we expressed E4ORF1 of Ad36 or Ad5 or fluorescent tag alone by using recombinant adeno-associated viral vector in the liver of three mouse models. In db/db or diet-induced obesity (DIO) mice, hepatic expression of Ad36E4ORF1 but not Ad5E4ORF1 robustly improved glycemic control. In normoglycemic wild-type mice, hepatic expression of Ad36E4ORF1 lowered nonfasting blood glucose at a high dose of expression. Of note, Ad36E4ORF1 significantly reduced insulin levels in db/db and DIO mice. The improvement in glycemic control was observed without stimulation of the proximal insulin signaling pathway. Collectively, these data indicate that Ad36E4ORF1 is not a typical sensitizer, mimetic, or secretagogue of insulin. Instead, it may have insulin-sparing action, which seems to reduce the need for insulin and, hence, to reduce insulin levels.

Genetic Manipulation of Brown Fat Via Oral Administration of an Engineered Recombinant Adeno-associated Viral Serotype Vector.

Recombinant adeno-associated virus (rAAV) vectors are attractive vehicles for gene therapy. Gene delivery to the adipose tissue using naturally occurring AAV serotypes is less successful compared to liver and muscle. Here, we demonstrate that oral administration of an engineered serotype Rec2 led to preferential transduction of brown fat with absence of transduction in the gastrointestinal track. Among the six natural and engineered serotypes being compared, Rec2 was the most efficient serotype achieving high level transduction at a dose 1~2 orders lower than reported doses for systemic administration. Overexpressing vascular endothelial growth factor (VEGF) in brown fat via oral administration of Rec2-VEGF vector increased the brown fat mass and enhanced thermogenesis. In contrast, knockdown VEGF in brown fat of VEGF (loxP) mice via Rec2-Cre vector hampered cold response and decreased brown fat mass. Oral administration of Rec2 vector provides a novel tool to genetically manipulate brown fat for research and therapeutic applications.