Adipocyte CD1d Gene Transfer Induces T Cell Expansion and Adipocyte Inflammation in CD1d Knockout Mice.

CD1d, a lipid Ag-presenting molecule for invariant NKT (iNKT) cells, is abundantly expressed on adipocytes and regulates adipose homeostasis through iNKT cells. CD1d gene expression was restored in visceral adipose tissue adipocytes of CD1d knockout (KO) mice to investigate the interactions between adipocytes and immune cells within adipose tissue. We developed an adipocyte-specific targeting recombinant adeno-associated viral vector, with minimal off-target transgene expression in the liver, to rescue CD1d gene expression in visceral adipose tissue adipocytes of CD1d KO mice, followed by assessment of immune cell alternations in adipose tissue and elucidation of the underlying mechanisms of alteration. We report that adeno-associated virus-mediated gene transfer of CD1d to adipocytes in CD1d KO mice fails to rescue iNKT cells but leads to massive and selective expansion of T cells within adipose tissue, particularly CD8+ T effector cells, that is associated with adipocyte NLRP3 inflammasome activation, dysregulation of adipocyte functional genes, and upregulation of apoptotic pathway proteins. An NLRP3 inhibitor has no effect on T cell phenotypes whereas depletion of CD8+ T cells significantly attenuates inflammasome activation and abolishes the dysregulation of adipocyte functional genes induced by adipocyte CD1d. In contrast, adipocyte overexpression of CD1d fails to induce T cell activation in wild-type mice or in invariant TCR α-chain Jα18 KO mice that have a normal lymphocyte repertoire except for iNKT cells. Our studies uncover an adipocyte CD1d → CD8+ T cell → adipocyte inflammasome cascade, in which CD8+ T cells function as a key mediator of adipocyte inflammation likely induced by an allogeneic response against the CD1d molecule.

Enriched environment enhances NK cell maturation through hypothalamic BDNF in male mice.

Macroenvironmental factors, including a patient's physical and social environment, play a role in cancer risk and progression. Our previous preclinical studies have shown that the enriched environment (EE) confers anti-obesity and anti-cancer phenotypes that are associated with enhanced adaptive immunity and are mediated by brain-derived neurotrophic factor (BDNF). Natural killer (NK) cells have anti-cancer and anti-viral properties, and their absence or depletion is associated with inferior clinical outcomes. In this study, we investigated the effects of EE on NK cell maturation following their depletion. Mice living in EE displayed a higher proportion of NK cells in the spleen, bone marrow, and blood, compared to those living in the standard environment (SE). EE enhanced NK cell maturation in the spleen and was associated with upregulation of BDNF expression in the hypothalamus. Hypothalamic BDNF overexpression reproduced the EE effects on NK cell maturation in secondary lymphoid tissues. Conversely, hypothalamic BDNF knockdown blocked the EE modulation on NK cell maturation. Our results demonstrate that a bio-behavior intervention enhanced NK cell maturation and was mediated at least in part by hypothalamic BDNF.

Environmental activation of a hypothalamic BDNF-adipocyte IL-15 axis regulates adipose-natural killer cells.

Physical and social environments influence immune homeostasis within adipose tissue, yet the mechanisms remain poorly defined. We report that an enriched environment (EE) housing modulates the immune cell population in white adipose tissue of mice including an increase in the abundance of natural killer (NK) cells. EE upregulates the expression of IL-15 and its receptor IL-15Rα specifically within mature adipocytes. Mechanistically, we show that hypothalamic brain-derived neurotrophic factor (BDNF) upregulates IL-15 production in adipocytes via sympathetic ß-adrenergic signaling. Overexpressing BDNF mediated by recombinant adeno-associated virus (rAAV) vector in the hypothalamus expands adipose NK cells. Conversely, inhibition of hypothalamic BDNF signaling via gene transfer of a dominant negative TrkB receptor suppresses adipose NK cells. In white adipose tissue, overexpression of IL-15 using an adipocyte-specific rAAV vector stimulates adipose NK cells and inhibits the progression of subcutaneous melanoma, whereas local IL-15 knockdown blocks the EE effect. These results suggest that bio-behavioral factors regulate adipose NK cells via a hypothalamic BDNF-sympathoneural-adipocyte IL-15 axis. Targeting this pathway may have therapeutic significance for cancer.

Adipocytes: A Novel Target for IL-15/IL-15Rα Cancer Gene Therapy.

IL-15 is a proinflammatory cytokine that plays an essential role in the development and activation of natural killer (NK) cells. Adipose tissue acts as an endocrine organ that secretes cytokines and is an important reservoir for lymphocytes. We hypothesized that activation of the IL-15 signaling in adipose tissue will activate and expand the NK cell population and control tumor growth. We recently developed an adipocyte-targeting recombinant adeno-associated viral (rAAV) vector with minimal off-target transgene expression in the liver. Here, we used this rAAV system to deliver an IL-15/IL-15Rα complex to the abdominal fat by intraperitoneal (i.p.) injection. Adipose IL-15/IL-15Rα complex gene transfer led to the expansion of NK cells in the adipose tissue and spleen in normal mice without notable side effects. The i.p. injection of rAAV-IL-15/IL-15Rα complex significantly suppressed the growth of Lewis lung carcinoma implanted subcutaneously and exerted a significant survival advantage in a B16-F10 melanoma metastasis model. The antitumor effects were associated with the expansion of the NK cells in the blood, spleen, abdominal fat, and tumor, as well as the enhancement of NK cell maturity. Our proof-of-concept preclinical studies demonstrate the safety and efficacy of the adipocyte-specific IL-15/IL-15Rα complex vector as a novel cancer immune gene therapy.

Enriched environment regulates thymocyte development and alleviates experimental autoimmune encephalomyelitis in mice.

Environmental and social factors have profound impacts on immune homeostasis. Our work on environmental enrichment (EE) has revealed a novel anti-obesity and anticancer phenotype associated with enhanced activity of CD8+ cytotoxic T lymphocytes in secondary lymphoid tissues. Here we investigated how an EE modulated thymus and thymocyte development. EE decreased thymus mass and cellularity, decreased the double positive thymocyte population, increased the proportion of CD8+ T cells, reduced the CD4:CD8 ratio, and downregulated CD69 expression in T cells. In a model of multiple sclerosis: experimental autoimmune encephalomyelitis (EAE), EE alleviated symptoms, inhibited spinal cord inflammation through regulation of type 1 T-helper cells mediated by glucocorticoid receptor signaling, and prevented EAE-induced thymic disturbance. Our mechanistic studies demonstrated that hypothalamic BDNF activated a hypothalamic-pituitary-adrenal axis mediating the EE's thymic effects. Our results indicate that a lifestyle intervention links the nervous, endocrine, and adaptive immune system, allowing the body to adapt to internal and external environments.

Environmental Enrichment Improves Motor Function and Muscle Transcriptome of Aged Mice.

Aging results in the progressive decline of muscle strength. Interventions to maintain muscle strength may mitigate the age-related loss of physical function, thus maximizing health span. The work on environmental enrichment (EE), an experimental paradigm recapitulating aspects of an active lifestyle, has revealed EE-induced metabolic benefits mediated by a brain-fat axis across the lifespan of mice. EE initiated at 18-month of age shows a trend toward an increased mean lifespan. While previous work described EE's influences on the aging dynamics of several central-peripheral processes, its influence on muscle remained understudied. Here, the impact of EE is investigated on motor function, neuromuscular physiology, and the skeletal muscle transcriptome. EE is initiated in 20-month-old mice for a five-month period. EE mice exhibit greater relative lean mass that is associated with improved mobility and hindlimb grip strength. Transcriptomic profiling of muscle tissue reveals an EE-associated enrichment of gene expression within several metabolic pathways related to oxidative phosphorylation and the TCA cycle. Many mitochondrial-related genes-several of which participate in the electron transport chain-are upregulated. Stress-responsive signaling pathways are downregulated because of EE. The results suggest that EE improves motor function-possibly through preservation of mitochondrial function-even late in life.

Protocol to minimize the confounding effect of cold stress on socially isolated mice using thermoneutral housing.

Social isolation, a risk factor for mortality and various disease states, in mice remains poorly understood, due in part to under-consideration of housing temperature and the murine thermoneutral zone. Here, we present a housing protocol to minimize the confounding effect of chronic cold stress on socially isolated mice that are unable to socially thermoregulate. We describe steps for allocating mice to group housing or social isolation conditions, housing mice in thermoneutral cabinets, feeding mice with high-fat diet, and measuring body weight, food intake, and metabolic indicators. For complete details on the use and execution of this protocol, please refer to Queen et al..1.

AAV-BDNF gene therapy ameliorates a hypothalamic neuroinflammatory signature in the Magel2-null model of Prader-Willi syndrome.

Individuals with Prader-Willi syndrome (PWS) exhibit several metabolic and behavioral abnormalities associated with excessive food-seeking activity. PWS is thought to be driven in part by dysfunctional hypothalamic circuitry and blunted responses to peripheral signals of satiety. Previous work described a hypothalamic transcriptomic signature of individuals with PWS. Notably, PWS patients exhibited downregulation of genes involved in neuronal development and an upregulation of neuroinflammatory genes. Deficiencies of brain-derived neurotrophic factor (BDNF) and its receptor were identified as potential drivers of PWS phenotypes. Our group recently applied an adeno-associated viral (AAV)-BDNF gene therapy within a preclinical PWS model, Magel2-null mice, to improve metabolic and behavioral function. While this proof-of-concept project was promising, it remained unclear how AAV-BDNF was influencing the hypothalamic microenvironment and how its therapeutic effect was mediated. To investigate, we hypothalamically injected AAV-BDNF to wild type and Magel2-null mice and performed mRNA sequencing on hypothalamic tissue. Here, we report that (1) Magel2 deficiency is associated with neuroinflammation in the hypothalamus and (2) AAV-BDNF gene therapy reverses this neuroinflammation. These data newly reveal Magel2-null mice as a valid model of PWS-related neuroinflammation and furthermore suggest that AAV-BDNF may modulate obesity-related neuroinflammatory phenotypes through direct or indirect means.

Social isolation exacerbates diet-induced obesity and peripheral inflammation in young male mice under thermoneutrality.

Social isolation (SI) is associated with an increased risk of mortality and various chronic diseases-including obesity-in humans. Murine studies probing SI metabolic outcomes remain inconsistent, due in part to a lack of consideration for housing temperature. Such experiments typically occur at room temperature, subjecting mice to chronic cold stress. Single housing prevents social thermoregulation, further exacerbating cold stress and obscuring psychosocial influences on metabolism at room temperature. In this study, C57BL/6 and BALB/c male mice were group- and single-housed under thermoneutral conditions to determine whether SI affects the development of high-fat diet-induced obesity. We report SI promotes weight gain, increases food intake, increases adiposity, worsens glycemic control, reduces insulin signaling, exacerbates systemic and adipose inflammatory responses, and induces a molecular signature within the hypothalamus. This study establishes a murine model that recapitulates the SI-induced propensity for obesity, which may further our understanding of SI's influence on health and disease.

Hypothalamic AAV-BDNF gene therapy improves metabolic function and behavior in the Magel2-null mouse model of Prader-Willi syndrome.

Individuals with Prader-Willi syndrome (PWS) display developmental delays, cognitive impairment, excessive hunger, obesity, and various behavioral abnormalities. Current PWS treatments are limited to strict supervision of food intake and growth hormone therapy, highlighting the need for new therapeutic strategies. Brain-derived neurotrophic factor (BDNF) functions downstream of hypothalamic feeding circuitry and has roles in energy homeostasis and behavior. In this preclinical study, we assessed the translational potential of hypothalamic adeno-associated virus (AAV)-BDNF gene therapy as a therapeutic for metabolic dysfunction in the Magel2-null mouse model of PWS. To facilitate clinical translation, our BDNF vector included an autoregulatory element allowing for transgene titration in response to the host's physiological needs. Hypothalamic BDNF gene transfer prevented weight gain, decreased fat mass, increased lean mass, and increased relative energy expenditure in female Magel2-null mice. Moreover, BDNF gene therapy improved glucose metabolism, insulin sensitivity, and circulating adipokine levels. Metabolic improvements were maintained through 23 weeks with no adverse behavioral effects, indicating high levels of efficacy and safety. Male Magel2-null mice also responded positively to BDNF gene therapy, displaying improved body composition, insulin sensitivity, and glucose metabolism. Together, these data suggest that regulating hypothalamic BDNF could be effective in the treatment of PWS-related metabolic abnormalities.

Hypothalamic gene transfer of BDNF promotes healthy aging.

The aging process and age-related diseases all involve metabolic decline and impaired ability to cope with adversity. Environmental enrichment (EE)-a housing environment which recapitulates aspects of active lifestyle-exerts a wide range of health benefits in laboratory rodents. Brain-derived neurotrophic factor (BDNF) in the hypothalamus orchestrates autonomic and neuroendocrine processes, serving as one key brain mediator of EE-induced resistance to obesity, cancer, and autoimmunity. Recombinant adeno-associated virus (AAV)-mediated hypothalamic BDNF gene transfer alleviates obesity, diabetes, and metabolic syndromes in both diet-induced and genetic models. One recent study by our lab demonstrates the efficacy and safety of a built-in autoregulatory system to control transgene BDNF expression, mimicking the body's natural feedback systems in middle-age mice. Twelve-month old mice were treated with autoregulatory BDNF vector and monitored for 7months. BDNF gene transfer prevented age-associated metabolic decline by: reducing adiposity, preventing the decline of brown fat activity, increasing adiponectin while reducing leptin and insulin in circulation, improving glucose tolerance, increasing energy expenditure, alleviating hepatic steatosis, and suppressing inflammatory genes in the hypothalamus and adipose tissues. Furthermore, BDNF treatment reduced anxiety-like and depression-like behaviors. This chapter summarizes this work and discusses potential roles that hypothalamic BDNF might play in promoting healthy aging.

Visceral adipose tissue-directed FGF21 gene therapy improves metabolic and immune health in BTBR mice.

Fibroblast growth factor 21 (FGF21) is a peptide hormone that serves as a potent effector of energy homeostasis. Increasingly, FGF21 is viewed as a promising therapeutic agent for type 2 diabetes, fatty liver disease, and other metabolic complications. Exogenous administration of native FGF21 peptide has proved difficult due to unfavorable pharmacokinetic properties. Here, we utilized an engineered serotype adeno-associated viral (AAV) vector coupled with a dual-cassette design to selectively overexpress FGF21 in visceral adipose tissue of insulin-resistant BTBR T+Itpr3tf/J (BTBR) mice. Under high-fat diet conditions, a single, low-dose intraperitoneal injection of AAV-FGF21 resulted in sustained benefits, including improved insulin sensitivity, glycemic processing, and systemic metabolic function and reduced whole-body adiposity, hepatic steatosis, inflammatory cytokines, and adipose tissue macrophage inflammation. Our study highlights the potential of adipose tissue as a FGF21 gene-therapy target and the promise of minimally invasive AAV vectors as therapeutic agents for metabolic diseases.

Environmental Enrichment Mitigates Age-Related Metabolic Decline and Lewis Lung Carcinoma Growth in Aged Female Mice.

Aging is a complex physiological process that leads to the progressive decline of metabolic and immune function, among other biological mechanisms. As global life expectancy increases, it is important to understand determinants of healthy aging-including environmental and genetic factors-and thus slow the onset or progression of age-related disease. Environmental enrichment (EE) is a housing environment wherein laboratory animals engage with complex physical and social stimulation. EE is a prime model to understand environmental influences on aging dynamics, as it confers an antiobesity and anticancer phenotype that has been implicated in healthy aging and health span extension. Although EE is frequently used to study malignancies in young mice, fewer studies characterize EE-cancer outcomes in older mice. Here, we used young (3-month-old) and aged (14-month-old) female C57BL/6 mice to determine whether EE would be able to mitigate age-related deficiencies in metabolic function and thus alter Lewis lung carcinoma (LLC) growth. Overall, EE improved metabolic function, resulting in reduced fat mass, increased lean mass, and improved glycemic processing; many of these effects were stronger in the aged cohort than in the young cohort, indicating an age-driven effect on metabolic responses. In the aged-EE cohort, subcutaneously implanted LLC tumor growth was inhibited and tumors exhibited alterations in various markers of apoptosis, proliferation, angiogenesis, inflammation, and malignancy. These results validate EE as an anticancer model in aged mice and underscore the importance of understanding environmental influences on cancer malignancy in aged populations. PREVENTION RELEVANCE: Environmental enrichment (EE) serves as a model of complex physical and social stimulation. This study validates EE as an anticancer intervention paradigm in aged mice and underscores the importance of understanding environmental influences on cancer malignancy in aged populations.

Improvements to Healthspan Through Environmental Enrichment and Lifestyle Interventions: Where Are We Now?

Environmental enrichment (EE) is an experimental paradigm that is used to explore how a complex, stimulating environment can impact overall health. In laboratory animal experiments, EE housing conditions typically include larger-than-standard cages, abundant bedding, running wheels, mazes, toys, and shelters which are rearranged regularly to further increase stimulation. EE has been shown to improve multiple aspects of health, including but not limited to metabolism, learning and cognition, anxiety and depression, and immunocompetence. Recent advances in lifespan have led some researchers to consider aging as a risk factor for disease. As such, there is a pressing need to understand the processes by which healthspan can be increased. The natural and predictable changes during aging can be reversed or decreased through EE and its underlying mechanisms. Here, we review the use of EE in laboratory animals to understand mechanisms involved in aging, and comment on relative areas of strength and weakness in the current literature. We additionally address current efforts toward applying EE-like lifestyle interventions to human health to extend healthspan. Although increasing lifespan is a clear goal of medical research, improving the quality of this added time also deserves significant attention. Despite hurdles in translating experimental results toward clinical application, we argue there is great potential in using features of EE toward improving human healthy life expectancy or healthspan, especially in the context of increased global longevity.

Regulation of aging and cancer by enhanced environmental activation of a hypothalamic-sympathoneural-adipocyte axis.

Social and environmental factors impact cancer and energy balance profoundly. Years ago, our lab established the existence of a novel brain-fat interaction we termed the "hypothalamic-sympathoneural-adipocyte (HSA) axis", through which complex environmental stimuli provided by an enriched environment regulate body composition, energy balance, and development of cancer. We have spent a significant portion of the past decade to further characterize the broad health benefits of an enriched environment (for example, leanness, enhanced immune function, and cancer resistance), and to identify mediators in the brain and periphery along the HSA axis. This review summarizes our recent work regarding the interface between endocrinology, immunology, cancer biology, aging, and neuroscience. We will discuss the interplay between these systemic phenomena and how the HSA axis can be targeted for regulation of cancer and aging.

Adipose PTEN acts as a downstream mediator of a brain-fat axis in environmental enrichment.

Background/Objectives: Environmental enrichment (EE) is a physiological model to investigate brain-fat interactions. We previously discovered that EE activates the hypothalamic-sympathoneural adipocyte (HSA) axis via induction of brain-derived neurotrophic factor (BDNF), thus leading to sympathetic stimulation of white adipose tissue (WAT) and an anti-obesity phenotype. Here, we investigate whether PTEN acts as a downstream mediator of the HSA axis in the EE. Methods: Mice were housed in EE for 4- and 16-week periods to determine how EE regulates adipose PTEN. Hypothalamic injections of adeno-associated viral (AAV) vectors expressing BDNF and a dominant negative form of its receptor were performed to assess the role of the HSA axis in adipose PTEN upregulation. A ß-blocker, propranolol, and a denervation agent, 6-hydroydopamine, were administered to assess sympathetic signaling in the observed EE-PTEN phenotype. To determine whether inducing PTEN is sufficient to reproduce certain EE adipose remodeling, we overexpressed PTEN in WAT using an AAV vector. To determine whether adipose PTEN is necessary for the EE-mediated reduction in adipocyte size, we injected a rAAV vector expressing Cre recombinase to the WAT of adult PTENflox mice and placed the mice in EE. Results: EE upregulated adipose PTEN expression, which was associated with suppression of AKT and ERK phosphorylation, increased hormone-sensitive lipase (HSL) phosphorylation, and reduced adiposity. PTEN regulation was found to be controlled by the HSA axis-with the hypothalamic BDNF acting as the upstream mediator-and dependent on sympathetic innervation. AAV-mediated adipose PTEN overexpression recapitulated EE-mediated adipose changes including suppression of AKT and ERK phosphorylation, increased HSL phosphorylation, and reduced adipose mass, whereas PTEN knockdown blocked the EE-induced reduction of adipocyte size. Conclusions: These data suggest that adipose PTEN responds to environmental stimuli and serves as downstream mediator of WAT remodeling in the EE paradigm, resulting in decreased adipose mass and decreased adipocyte size.

Environmental enrichment improves metabolic and behavioral health in the BTBR mouse model of autism.

BTBR T + Itpr3tf/J (BTBR) mice are an Autism Spectrum Disorder (ASD)-like model that exhibit behavioral and physiological deficits similar to those observed in patients with ASD. While behavioral therapy is a first line of treatment in ASD patients, comparable non-pharmacological treatments are less explored in murine models. Here, we administer a bio-behavioral intervention for BTBR mice by way of environmental enrichment (EE) - an experimental housing paradigm previously shown to improve systemic metabolism, learning/memory, anxious behavior, neurogenesis, locomotion, and immunocompetence in C57BL/6 mice. Juvenile BTBR mice were randomized to standard or EE housing and were subjected to metabolic and behavioral assessments up to 17 weeks. Following EE exposure, we report an EE-induced metabolic and behavioral phenotype. Male BTBR mice responded metabolically to EE, displaying reduced adiposity, increased lean mass, improved glycemic control, and decreased circulating leptin. The gene expressions of brain-derived neurotrophic factor (Bdnf) and its receptor (Ntrk2/TrkB) were upregulated in several brain areas in EE-BTBR males. EE-BTBR females showed modest reduction of adiposity and no changes in glycemic control, circulating leptin, or Bdnf/Ntrk2 gene expression. With regard to behavior, EE resulted in decreased anxiety, and increased social affiliation. Together, these results suggest that EE improves metabolic and behavioral health in BTBR mice.

CSF1R inhibitor PLX5622 and environmental enrichment additively improve metabolic outcomes in middle-aged female mice.

As the elderly population grows, chronic metabolic dysfunction including obesity and diabetes are becoming increasingly common comorbidities. Hypothalamic inflammation through CNS resident microglia serves as a common pathway between developing obesity and developing systemic aging pathologies. Despite understanding aging as a life-long process involving interactions between individuals and their environment, limited studies address the dynamics of environment interactions with aging or aging therapeutics. We previously demonstrated environmental enrichment (EE) is an effective model for studying improved metabolic health and overall healthspan in mice, which acts through a brain-fat axis. Here we investigated the CSF1R inhibitor PLX5622 (PLX), which depletes microglia, and its effects on metabolic decline in aging in interaction with EE. PLX in combination with EE substantially improved metabolic outcomes in middle-aged female mice over PLX or EE alone. Chronic PLX treatment depleted 75% of microglia from the hypothalamus and reduced markers of inflammation without affecting brain-derived neurotrophic factor levels induced by EE. Adipose tissue remodeling and adipose tissue macrophage modulation were observed in response to CSF1R inhibition, which may contribute to the combined benefits seen in EE with PLX. Our study suggests benefits exist from combined drug and lifestyle interventions in aged animals.

rAAV-Mediated Gene Delivery to Adipose Tissue.

Recombinant adeno-associated virus (rAAV) vectors are attractive vehicles for gene therapy. Yet, it is challenging to genetically manipulate adipose tissue in adults due to the low transduction efficiency of naturally occurring AAV serotypes. We recently demonstrated that a novel engineered hybrid serotype Rec2 achieves high transduction of adipose tissue that is superior to naturally occurring serotypes via direct injection to adipose depots. Furthermore, the administration route influences the tropism and efficacy of Rec2 vector: oral administration transduces interscapular brown fat, while intraperitoneal injection preferentially targets visceral fat. Multiple in vivo studies by our lab and others have demonstrated that Rec2 vector provides a powerful tool to genetically manipulate adipose tissue for basic research and potential gene therapies of genetic and acquired diseases. Here we provide detailed protocols for AAV production and delivery to adipose tissue by direct injection, oral administration, and intraperitoneal injection.

Adipose PTEN regulates adult adipose tissue homeostasis and redistribution via a PTEN-leptin-sympathetic loop.

OBJECTIVE: Despite the large body of work describing the tumor suppressor functions of Phosphatase and tensin homologue deleted on chromosome ten (PTEN), its roles in adipose homeostasis of adult animals are not yet fully understood. Here, we sought to determine the role of PTEN in whole-body adipose homeostasis. METHODS: We genetically manipulated PTEN in specific fat depots through recombinant adeno-associated viral vector (rAAV)-based gene transfer of Cre recombinase to adult PTENflox mice. Additionally, we used a denervation agent, 6OHDA, to assess the role of sympathetic signaling in PTEN-related adipose remodeling. Furthermore, we chemically manipulated AKT signaling via a pan-AKT inhibitor, MK-2206, to assess the role of AKT in PTEN-related adipose remodeling. Finally, to understand the role of leptin and central signaling on peripheral tissues, we knocked down hypothalamic leptin receptor with a microRNA delivered by a rAAV vector. RESULTS: Knockdown PTEN in individual fat depot resulted in massive expansion of the affected fat depot through activation of AKT signaling associated with suppression of lipolysis and induction of leptin. This hypertrophic expansion of the affected fat depot led to upregulation of PTEN level, higher lipolysis, and induction of white fat browning in other fat depots, and the compensatory reduced fat mass to maintain a set point of whole-body adiposity. Administration of AKT inhibitor MK-2206 prevented the adipose PTEN knockdown-associated effects. 6OHDA-mediated denervation demonstrated that sympathetic innervation was required for the PTEN knockdown-induced adipose redistribution. Knockdown hypothalamic leptin receptor attenuated the adipose redistribution induced by PTEN deficiency in individual fat depot. CONCLUSIONS: Our results demonstrate the essential role of PTEN in adipose homeostasis, including mass and distribution in adulthood, and reveal an "adipose PTEN-leptin-sympathetic nervous system" feedback loop to maintain a set point of adipose PTEN and whole-body adiposity.