Brown adipose tissue whitening leads to brown adipocyte death and adipose tissue inflammation.

In mammals, white adipose tissue (WAT) stores and releases lipids, whereas brown adipose tissue (BAT) oxidizes lipids to fuel thermogenesis. In obese individuals, WAT undergoes profound changes; it expands, becomes dysfunctional, and develops a low-grade inflammatory state. Importantly, BAT content and activity decline in obese subjects, mainly as a result of the conversion of brown adipocytes to white-like unilocular cells. Here, we show that BAT "whitening" is induced by multiple factors, including high ambient temperature, leptin receptor deficiency, ß-adrenergic signaling impairment, and lipase deficiency, each of which is capable of inducing macrophage infiltration, brown adipocyte death, and crown-like structure (CLS) formation. Brown-to-white conversion and increased CLS formation were most marked in BAT from adipose triglyceride lipase (Atgl)-deficient mice, where, according to transmission electron microscopy, whitened brown adipocytes contained enlarged endoplasmic reticulum, cholesterol crystals, and some degenerating mitochondria, and were surrounded by an increased number of collagen fibrils. Gene expression analysis showed that BAT whitening in Atgl-deficient mice was associated to a strong inflammatory response and NLRP3 inflammasome activation. Altogether, the present findings suggest that converted enlarged brown adipocytes are highly prone to death, which, by promoting inflammation in whitened BAT, may contribute to the typical inflammatory state seen in obesity.

Fto-Deficiency Affects the Gene and MicroRNA Expression Involved in Brown Adipogenesis and Browning of White Adipose Tissue in Mice.

Genetic variants in the fat mass- and obesity-associated gene Fto are linked to the onset of obesity in humans. The causal role of the FTO protein in obesity is supported by evidence obtained from transgenic mice; however, the underlying molecular pathways pertaining to the role of FTO in obesity have yet to be established. In this study, we investigate the Fto gene in mouse brown adipose tissue and in the browning process of white adipose tissue. We analyze distinct structural and molecular factors in brown and white fat depots of Fto-deficient mice under normal and obesogenic conditions. We report significant alterations in the morphology of adipose tissue depots and the expression of mRNA and microRNA related to brown adipogenesis and metabolism in Fto-deficient mice. Furthermore, we show that high-fat feeding does not attenuate the browning process of Fto-deficient white adipose tissue as observed in wild-type tissue, suggesting a triggering effect of the FTO pathways by the dietary environment.

Adipo-Epithelial Transdifferentiation in In Vitro Models of the Mammary Gland.

Subcutaneous adipocytes are crucial for mammary gland epithelial development during pregnancy. Our and others' previous data have suggested that adipo-epithelial transdifferentiation could play a key role in the mammary gland alveolar development. In this study, we tested whether adipo-epithelial transdifferentiation occurs in vitro. Data show that, under appropriate co-culture conditions with mammary epithelial organoids (MEOs), mature adipocytes lose their phenotype and acquire an epithelial one. Interestingly, even in the absence of MEOs, extracellular matrix and diffusible growth factors are able to promote adipo-epithelial transdifferentiation. Gene and protein expression studies indicate that transdifferentiating adipocytes exhibit some characteristics of milk-secreting alveolar glands, including significantly higher expression of milk proteins such as whey acidic protein and ß-casein. Similar data were also obtained in cultured human multipotent adipose-derived stem cell adipocytes. A miRNA sequencing experiment on the supernatant highlighted mir200c, which has a well-established role in the mesenchymal-epithelial transition, as a potential player in this phenomenon. Collectively, our data show that adipo-epithelial transdifferentiation can be reproduced in in vitro models where this phenomenon can be investigated at the molecular level.

Obese adipocytes show ultrastructural features of stressed cells and die of pyroptosis.

We previously suggested that, in obese animals and humans, white adipose tissue inflammation results from the death of hypertrophic adipocytes; these are then cleared by macrophages, giving rise to distinctive structures we denominated crown-like structures. Here we present evidence that subcutaneous and visceral hypertrophic adipocytes of leptin-deficient (ob/ob and db/db) obese mice exhibit ultrastructural abnormalities (including calcium accumulation and cholesterol crystals), many of which are more common in hyperglycemic db/db versus normoglycemic ob/ob mice and in visceral versus subcutaneous depots. Degenerating adipocytes whose intracellular content disperses in the extracellular space were also noted in obese mice; in addition, increased anti-reactive oxygen species enzyme expression in obese fat pads, documented by RT-PCR and immunohistochemistry, suggests that ultrastructural changes are accompanied by oxidative stress. RT-PCR showed NLRP3 inflammasome activation in the fat pads of both leptin-deficient and high-fat diet obese mice, in which formation of active caspase-1 was documented by immunohistochemistry in the cytoplasm of several hypertrophic adipocytes. Notably, caspase-1 was not detected in FAT-ATTAC transgenic mice, where adipocytes die of apoptosis. Thus, white adipocyte overexpansion induces a stress state that ultimately leads to death. NLRP3-dependent caspase-1 activation in hypertrophic adipocytes likely induces obese adipocyte death by pyroptosis, a proinflammatory programmed cell death.

Mesenchymal stromal cells from human umbilical cord prevent the development of lung fibrosis in immunocompetent mice.

Lung fibrosis is a severe condition resulting from several interstial lung diseases (ILD) with different etiologies. Current therapy of ILD, especially those associated with connective tissue diseases, is rather limited and new anti-fibrotic strategies are needed. In this study, we investigated the anti-fibrotic activity in vivo of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC). Adult immunocompetent C57BL/6 mice (n. = 8 for each experimental condition) were injected intravenously with hUC-MSC (n. = 2.5 × 105) twice, 24 hours and 7 days after endotracheal injection of bleomycin. Upon sacrifice at days 8, 14, 21, collagen content, inflammatory cytokine profile, and hUC-MSC presence in explanted lung tissue were analyzed. Systemic administration of a double dose of hUC-MSC significantly reduced bleomycin-induced lung injury (inflammation and fibrosis) in mice through a selective inhibition of the IL6-IL10-TGFß axis involving lung M2 macrophages. Only few hUC-MSC were detected from explanted lungs, suggesting a "hit and run" mechanism of action of this cellular therapy. Our data indicate that hUC-MSC possess strong in vivo anti-fibrotic activity in a mouse model resembling an immunocompetent human subject affected by inflammatory ILD, providing proof of concept for ad-hoc clinical trials.

Stress-induced activation of brown adipose tissue prevents obesity in conditions of low adaptive thermogenesis.

BACKGROUND: Stress-associated conditions such as psychoemotional reactivity and depression have been paradoxically linked to either weight gain or weight loss. This bi-directional effect of stress is not understood at the functional level. Here we tested the hypothesis that pre-stress level of adaptive thermogenesis and brown adipose tissue (BAT) functions explain the vulnerability or resilience to stress-induced obesity. METHODS: We used wt and triple ß1,ß2,ß3-Adrenergic Receptors knockout (ß-less) mice exposed to a model of chronic subordination stress (CSS) at either room temperature (22 °C) or murine thermoneutrality (30 °C). A combined behavioral, physiological, molecular, and immunohistochemical analysis was conducted to determine stress-induced modulation of energy balance and BAT structure and function. Immortalized brown adipocytes were used for in vitro assays. RESULTS: Departing from our initial observation that ßARs are dispensable for cold-induced BAT browning, we demonstrated that under physiological conditions promoting low adaptive thermogenesis and BAT activity (e.g. thermoneutrality or genetic deletion of the ßARs), exposure to CSS acted as a stimulus for BAT activation and thermogenesis, resulting in resistance to diet-induced obesity despite the presence of hyperphagia. Conversely, in wt mice acclimatized to room temperature, and therefore characterized by sustained BAT function, exposure to CSS increased vulnerability to obesity. Exposure to CSS enhanced the sympathetic innervation of BAT in wt acclimatized to thermoneutrality and in ß-less mice. Despite increased sympathetic innervation suggesting adrenergic-mediated browning, norepinephrine did not promote browning in ßARs knockout brown adipocytes, which led us to identify an alternative sympathetic/brown adipocytes purinergic pathway in the BAT. This pathway is downregulated under conditions of low adaptive thermogenesis requirements, is induced by stress, and elicits activation of UCP1 in wt and ß-less brown adipocytes. Importantly, this purinergic pathway is conserved in human BAT. CONCLUSION: Our findings demonstrate that thermogenesis and BAT function are determinant of the resilience or vulnerability to stress-induced obesity. Our data support a model in which adrenergic and purinergic pathways exert complementary/synergistic functions in BAT, thus suggesting an alternative to ßARs agonists for the activation of human BAT.

Activation of transcription factors STAT1 and STAT5 in the mouse median eminence after systemic ciliary neurotrophic factor administration.

Exogenously administered ciliary neurotrophic factor (CNTF) causes weight loss in obese rodents and humans through leptin-like activation of the Jak-STAT3 signaling pathway in hypothalamic arcuate neurons. Here we report for the first time that 40min after acute systemic treatment, rat recombinant CNTF (intraperitoneal injection of 0.3mg/kg of body weight) induced nuclear translocation of the tyrosine-phosphorylated forms of STAT1 and STAT5 in the mouse median eminence and other circumventricular organs, including the vascular organ of the lamina terminalis and the subfornical organ. In the tuberal hypothalamus of treated mice, specific nuclear immunostaining for phospo-STAT1 and phospho-STAT5 was detected in ependymal cells bordering the third ventricle floor and lateral recesses, and in median eminence cells. Co-localization studies documented STAT1 and STAT5 activation in median eminence ß-tanycytes and underlying radial glia-like cells. A few astrocytes in the arcuate nucleus responded to CNTF by STAT5 activation. The vast majority of median eminence tanycytes and radial glia-like cells showing phospho-STAT1 and phospho-STAT5 immunoreactivity were also positive for phospho-STAT3. In contrast, STAT3 was the sole STAT isoform activated by CNTF in arcuate nucleus and median eminence neurons. Finally, immunohistochemical evaluation of STAT activation 20, 40, 80, and 120min from the injection demonstrated that cell activation was accompanied by c-Fos expression. Collectively, our findings show that CNTF activates STAT3, STAT1, and STAT5 in vivo. The distinctive activation pattern of these STAT isoforms in the median eminence may disclose novel targets and pathways through which CNTF regulates food intake.

Glial-like differentiation potential of human mature adipocytes.

The potential ability to differentiate dedifferentiated adipocytes into a neural lineage is attracting strong interest as an emerging method of producing model cells for the treatment of a variety of neurological diseases. Here, we describe the efficient conversion of dedifferentiated adipocytes into a neural-like cell population. These cells grew in neurosphere-like structures and expressed a high level of the early neuroectodermal marker Nestin. These neurospheres could proliferate and express stemness genes, suggesting that these cells could be committed to the neural lineage. After neural induction, NeuroD1, Sox1, Double Cortin, and Eno2 were not expressed. Patch clamp data did not reveal different electrophysiological properties, indicating the inability of these cells to differentiate into mature neurons. In contrast, the differentiated cells expressed a high level of CLDN11, as demonstrated using molecular method, and stained positively for the glial cell markers CLDN11 and GFAP, as demonstrated using immunocytochemistry. These data were confirmed by quantitative results for glial cell line-derived neurotrophic factor production, which showed a higher secretion level in neurospheres and the differentiated cells compared with the untreated cells. In conclusion, our data demonstrate morphological, molecular, and immunocytochemical evidence of initial neural differentiation of mature adipocytes, committing to a glial lineage.

Plasticity of human dedifferentiated adipocytes toward endothelial cells.

The process of cellular differentiation in terminally differentiated cells is thought to be irreversible, and these cells are thought to be incapable of differentiating into distinct cell lineages. Our previous study showed that mature adipocytes represent an alternative source of mesenchymal stem cells. Here, results showed the capacity of mature adipocytes to differentiate into endothelial-like cells, using the ability of these cells to revert into an immature phase without any relievable chromosomal alterations. Mature adipocytes were isolated from human omental and subcutaneous fat and were dedifferentiated in vitro. The resulting cells were subcultivated for endothelial differentiation and were analyzed for their expression of specific genes and proteins. Endothelial-like cells were harvested from the differentiation medium and were traditionally cultured to evaluate the endothelial markers and the karyotype. Cells cultured in specific medium formed tube-like structures and expressed several endothelial marker genes and proteins. The endothelial-like cells expressed significantly higher levels of vascular endothelium growth factor receptor 2, vascular endothelial cadherin, Von Willebrand factor, and CD133 than the untreated cells. These cells were positively stained for CD31 and vascular endothelial cadherin, markers of mature endothelial cells. Moreover, the low-density lipoprotein-uptake assay demonstrated a functionally endothelial differentiation of these cells. When these cells were harvested and reseeded in basal medium, they lost the endothelial markers and reacquired the typical mesenchymal stem cell markers and the ability to expand in a short time period. Moreover, karyotype analysis showed that these cells reverted into an immature phase without any karyotype alterations. In conclusion, the results showed that adipocytes exhibited a great plasticity toward the endothelial lineage, suggesting their possible use in cell therapy applications for vascular disease.

Fat mass- and obesity-associated gene Fto affects the dietary response in mouse white adipose tissue.

Common variants of human fat mass- and obesity-associated gene Fto have been linked with higher body mass index, but the biological explanation for the link has remained obscure. Recent findings suggest that these variants affect the homeobox protein IRX3. Here we report that FTO has a role in white adipose tissue which modifies its response to high-fat feeding. Wild type and Fto-deficient mice were exposed to standard or high-fat diet for 16 weeks after which metabolism, behavior and white adipose tissue morphology were analyzed together with adipokine levels and relative expression of genes regulating white adipose tissue adipogenesis and Irx3. Our results indicate that Fto deficiency increases the expression of genes related to adipogenesis preventing adipocytes from becoming hypertrophic after high-fat diet. In addition, we report a novel finding of increased Irx3 expression in Fto-deficient mice after high-fat feeding indicating a complex link between FTO, IRX3 and fat metabolism.

Opposite effects of a high-fat diet and calorie restriction on ciliary neurotrophic factor signaling in the mouse hypothalamus.

In the mouse hypothalamus, ciliary neurotrophic factor (CNTF) is mainly expressed by ependymal cells and tanycytes of the ependymal layer covering the third ventricle. Since exogenously administered CNTF causes reduced food intake and weight loss, we tested whether endogenous CNTF might be involved in energy balance regulation. We thus evaluated CNTF production and responsiveness in the hypothalamus of mice fed a high-fat diet (HFD), of ob/ob obese mice, and of mice fed a calorie restriction (CR) regimen. RT-PCR showed that CNTF mRNA increased significantly in HFD mice and decreased significantly in CR animals. Western blotting confirmed that CNTF expression was higher in HFD mice and reduced in CR mice, but high interindividual variability blunted the significance of these differences. By immunohistochemistry, hypothalamic tuberal and mammillary region tanycytes stained strongly for CNTF in HFD mice, whereas CR mice exhibited markedly reduced staining. RT-PCR and Western blotting disclosed that changes in CNTF expression were paralleled by changes in the expression of its specific receptor, CNTF receptor α (CNTFRα). Injection of recombinant CNTF and detection of phospho-signal transducer and activator of transcription 3 (P-STAT3) showed that CNTF responsiveness by the ependymal layer, mainly by tanycytes, was higher in HFD than CR mice. In addition, in HFD mice CNTF administration induced distinctive STAT3 signaling in a large neuron population located in the dorsomedial and ventromedial nuclei, perifornical area and mammillary body. The hypothalamic expression of CNTF and CNTFRα did not change in the hyperphagic, leptin-deficient ob/ob obese mice; accordingly, P-STAT3 immunoreactivity in CNTF-treated ob/ob mice was confined to ependymal layer and arcuate neurons. Collectively, these data suggest that hypothalamic CNTF is involved in controlling the energy balance and that CNTF signaling plays a role in HFD obese mice at specific sites.