FASN deficiency induces a cytosol-to-mitochondria citrate flux to mitigate detachment-induced oxidative stress.

Fatty acid synthase (FASN) maintains de novo lipogenesis (DNL) to support rapid growth in most proliferating cancer cells. Lipogenic acetyl-coenzyme A (CoA) is primarily produced from carbohydrates but can arise from glutamine-dependent reductive carboxylation. Here, we show that reductive carboxylation also occurs in the absence of DNL. In FASN-deficient cells, reductive carboxylation is mainly catalyzed by isocitrate dehydrogenase-1 (IDH1), but IDH1-generated cytosolic citrate is not utilized for supplying DNL. Metabolic flux analysis (MFA) shows that FASN deficiency induces a net cytosol-to-mitochondria citrate flux through mitochondrial citrate transport protein (CTP). Previously, a similar pathway has been shown to mitigate detachment-induced oxidative stress in anchorage-independent tumor spheroids. We further report that tumor spheroids show reduced FASN activity and that FASN-deficient cells acquire resistance to oxidative stress in a CTP- and IDH1-dependent manner. Collectively, these data indicate that by inducing a cytosol-to-mitochondria citrate flux, anchorage-independent malignant cells can gain redox capacity by trading off FASN-supported rapid growth.

FASN-deficiency induces a cytosol-to-mitochondria citrate flux to mitigate detachment-induced oxidative stress.

Fatty acid synthase (FASN) maintains de novo lipogenesis (DNL) to support rapid growth in most proliferating cancer cells. Lipogenic acetyl-CoA is primarily produced from carbohydrates but can arise from glutamine-dependent reductive carboxylation under hypoxia. Here we show that reductive carboxylation also occurs in the absence of DNL in cells with defective FASN. In this state, reductive carboxylation was mainly catalyzed by isocitrate dehydrogenase-1 (IDH1) in the cytosol, but IDH1-generated citrate was not used for DNL. Metabolic flux analysis (MFA) revealed that FASN-deficiency induced a net cytosol-to-mitochondria citrate flux through citrate transport protein (CTP). A similar pathway was previously shown to mitigate detachment-induced mitochondrial reactive oxygen species (mtROS) in anchorage-independent tumor spheroids. We further demonstrate that FASN-deficient cells acquire resistance to oxidative stress in a CTP- and IDH1-dependent manner. Together with the reduced FASN activity in tumor spheroids, these data indicate that anchorage-independent malignant cells trade FASN-supported rapid growth for a cytosol-to-mitochondria citrate flux to gain redox capacity against detachment-induced oxidative stress.

Characterization of municipal solid waste incineration and flue gas emission under anoxic environment in Tibet Plateau.

Waste incineration is a process of full combustion reaction between waste and oxygen at high temperature. It is a new problem whether the special natural environmental conditions of Tibet Plateau, such as low air pressure, low oxygen content, and low temperature, will affect the waste incineration in the plateau area. In this work, the influence of different parameters on MSW incineration efficiency and flue gas emission were investigated. The results showed that the temperatures exhibited a significant impact on the flue gas emission. Under the lower temperature, CO was determined to be the main pollutant. With the increase of temperature, NOx became the main pollutant. The optimal temperature range of flue gas emission was between 800 and 900°C. A slight negative pressure in incinerator was benefit for incineration system safety and flue gas emissions. The optimal range was -50 to 0Pa. Lower oxygen content (3-6%) in the incinerator affected the incineration efficiency and flue gas emission. Meanwhile, the high oxygen content had no obvious impact on the flue gas emission, but the cost increased and the service life of the incinerator was affected. The optimal oxygen content in the incinerator was controlled at 6-8%. Furthermore, the air temperatures, pressures, and oxygen content in the natural environment had no significant effect on MSW incineration process. Increasing the air volume would bring about the increase of N2 in the incinerator. This work provides the basic data support for MSW incineration technology in plateau area.

The B56α subunit of PP2A is necessary for mesenchymal stem cell commitment to adipocyte.

Adipose tissue plays a major role in maintaining organismal metabolic equilibrium. Control over the fate decision from mesenchymal stem cells (MSCs) to adipocyte differentiation involves coordinated command of phosphorylation. Protein phosphatase 2A plays an important role in Wnt pathway and adipocyte development, yet how PP2A complexes actively respond to adipocyte differentiation signals and acquire specificity in the face of the promiscuous activity of its catalytic subunit remains unknown. Here, we report the PP2A phosphatase B subunit B56α is specifically induced during adipocyte differentiation and mediates PP2A to dephosphorylate GSK3ß, thereby blocking Wnt activity and driving adipocyte differentiation. Using an inducible B56α knock-out mouse, we further demonstrate that B56α is essential for gonadal adipose tissue development in vivo and required for the fate decision of adipocytes over osteoblasts. Moreover, we show B56α expression is driven by the adipocyte transcription factor PPARγ thereby establishing a novel link between PPARγ signaling and Wnt blockade. Overall, our results reveal B56α is a necessary part of the machinery dictating the transition from pre-adipocyte to mature adipocyte and provide fundamental insights into how PP2A complex specifically and actively regulates unique signaling pathway in biology.

Mitochondrial division inhibitor (mdivi-1) decreases oxidative metabolism in cancer.

BACKGROUND: Previous studies suggested that mdivi-1 (mitochondrial division inhibitor), a putative inhibitor of dynamin-related protein (DRP1), decreased cancer cell proliferation through inducing mitochondrial fusion and altering oxygen consumption. However, the metabolic reprogramming underlying the DRP1 inhibition is still unclear in cancer cells. METHODS: To better understand the metabolic effect of DRP1 inhibition, [U-13C]glucose isotope tracing was employed to assess mdivi-1 effects in several cancer cell lines, DRP1-WT (wild-type) and DRP1-KO (knockout) H460 lung cancer cells and mouse embryonic fibroblasts (MEFs). RESULTS: Mitochondrial staining confirmed that mdivi-1 treatment and DRP1 deficiency induced mitochondrial fusion. Surprisingly, metabolic isotope tracing found that mdivi-1 decreased mitochondrial oxidative metabolism in the lung cancer cell lines H460, A549 and the colon cancer cell line HCT116. [U-13C]glucose tracing studies also showed that the TCA cycle intermediates had significantly lower enrichment in mdivi-1-treated cells. In comparison, DRP1-WT and DRP1-KO H460 cells had similar oxidative metabolism, which was decreased by mdivi-1 treatment. Furthermore, mdivi-1-mediated effects on oxidative metabolism were independent of mitochondrial fusion. CONCLUSIONS: Our data suggest that, in cancer cells, mdivi-1, a putative inhibitor of DRP1, decreases oxidative metabolism to impair cell proliferation.

Remodeling of Murine Mammary Adipose Tissue during Pregnancy, Lactation, and Involution.

White adipocytes in the mammary gland stroma comprise the majority of the mammary gland mass. White adipocytes regulate numerous hormonal and metabolic processes and exhibit compositional and phenotypic plasticity. This plasticity is exemplified by the ability of mammary adipocytes to regress during lactation, when mammary epithelial cells expand to establish sufficient milk-producing alveoli. Upon weaning, the process reverses through mammary involution, during which adipocytes extensively regenerate, and alveolar epithelial cells disappear through cell death, returning the mammary gland to the non-lactating state. Despite intensive studies on the development and involution of the mammary alveolar epithelium, the fate of mammary adipocytes during pregnancy and lactation, and the origins of mammary adipocytes regenerated during mammary involution, is poorly understood. Here, we discuss the recent discoveries of the fate of mammary adipocytes during pregnancy and lactation in a number of different mouse models, and the lineage origin of mammary adipocytes regenerated during involution.

The correlation of copy number variations with longevity in a genome-wide association study of Han Chinese.

Copy number variations (CNVs) have been shown to cause numerous diseases, however, their roles in human lifespan remain elusive. In this study, we investigate the association of CNVs with longevity by comparing the Han Chinese genomes of long-lived individuals from 90 to 117 years of age and the middle-aged from 30 to 65. Our data demonstrate that the numbers of CNVs, especially deletions, increase significantly in a direct correlation with longevity. We identify eleven CNVs that strongly associate with longevity; four of them locate in the chromosome bands, 7p11.2, 20q13.33, 19p12 and 8p23.3 and overlap partially with the CNVs identified in long-lived Danish or U.S. populations, while the other seven have not been reported previously. These CNV regions encode nineteen known genes, and some of which have been shown to affect aging-related phenotypes such as the shortening of telomere length (ZNF208), the risk of cancer (FOXA1, LAMA5, ZNF716), and vascular and immune-related diseases (ARHGEF10, TOR2A, SH2D3C). In addition, we found several pathways enriched in long-lived genomes, including FOXA1 and FOXA transcription factor networks involved in regulating aging or age-dependent diseases such as cancer. Thus, our study has identified longevity-associated CNV regions and their affected genes and pathways. Our results suggest that the human genome structures such as CNVs might play an important role in determining a long life in human.

Reductive carboxylation supports redox homeostasis during anchorage-independent growth.

Cells receive growth and survival stimuli through their attachment to an extracellular matrix (ECM). Overcoming the addiction to ECM-induced signals is required for anchorage-independent growth, a property of most malignant cells. Detachment from ECM is associated with enhanced production of reactive oxygen species (ROS) owing to altered glucose metabolism. Here we identify an unconventional pathway that supports redox homeostasis and growth during adaptation to anchorage independence. We observed that detachment from monolayer culture and growth as anchorage-independent tumour spheroids was accompanied by changes in both glucose and glutamine metabolism. Specifically, oxidation of both nutrients was suppressed in spheroids, whereas reductive formation of citrate from glutamine was enhanced. Reductive glutamine metabolism was highly dependent on cytosolic isocitrate dehydrogenase-1 (IDH1), because the activity was suppressed in cells homozygous null for IDH1 or treated with an IDH1 inhibitor. This activity occurred in absence of hypoxia, a well-known inducer of reductive metabolism. Rather, IDH1 mitigated mitochondrial ROS in spheroids, and suppressing IDH1 reduced spheroid growth through a mechanism requiring mitochondrial ROS. Isotope tracing revealed that in spheroids, isocitrate/citrate produced reductively in the cytosol could enter the mitochondria and participate in oxidative metabolism, including oxidation by IDH2. This generates NADPH in the mitochondria, enabling cells to mitigate mitochondrial ROS and maximize growth. Neither IDH1 nor IDH2 was necessary for monolayer growth, but deleting either one enhanced mitochondrial ROS and reduced spheroid size, as did deletion of the mitochondrial citrate transporter protein. Together, the data indicate that adaptation to anchorage independence requires a fundamental change in citrate metabolism, initiated by IDH1-dependent reductive carboxylation and culminating in suppression of mitochondrial ROS.

Impact of tamoxifen on adipocyte lineage tracing: Inducer of adipogenesis and prolonged nuclear translocation of Cre recombinase.

BACKGROUND: The selective estrogen receptor modulator tamoxifen, in combination with the Cre-ER(T2) fusion protein, has been one of the mainstream methods to induce genetic recombination and has found widespread application in lineage tracing studies. METHODS & RESULTS: Here, we report that tamoxifen exposure at widely used concentrations remains detectable by mass-spectrometric analysis in adipose tissue after a washout period of 10 days. Surprisingly, its ability to maintain nuclear translocation of the Cre-ER(T2) protein is preserved beyond 2 months of washout. Tamoxifen treatment acutely leads to transient lipoatrophy, followed by de novo adipogenesis that reconstitutes the original fat mass. In addition, we find a "synthetically lethal" phenotype for adipocytes when tamoxifen treatment is combined with adipocyte-specific loss-of-function mutants, such as an adipocyte-specific PPARγ knockout. This is observed to a lesser extent when alternative inducible approaches are employed. CONCLUSIONS: These findings highlight the potential for tamoxifen-induced adipogenesis, and the associated drawbacks of the use of tamoxifen in lineage tracing studies, explaining the discrepancy in lineage tracing results from different systems with temporal control of gene targeting.

Adipocyte inflammation is essential for healthy adipose tissue expansion and remodeling.

Chronic inflammation constitutes an important link between obesity and its pathophysiological sequelae. In contrast to the belief that inflammatory signals exert a fundamentally negative impact on metabolism, we show that proinflammatory signaling in the adipocyte is in fact required for proper adipose tissue remodeling and expansion. Three mouse models with an adipose tissue-specific reduction in proinflammatory potential were generated that display a reduced capacity for adipogenesis in vivo, while the differentiation potential is unaltered in vitro. Upon high-fat-diet exposure, the expansion of visceral adipose tissue is prominently affected. This is associated with decreased intestinal barrier function, increased hepatic steatosis, and metabolic dysfunction. An impaired local proinflammatory response in the adipocyte leads to increased ectopic lipid accumulation, glucose intolerance, and systemic inflammation. Adipose tissue inflammation is therefore an adaptive response that enables safe storage of excess nutrients and contributes to a visceral depot barrier that effectively filters gut-derived endotoxin.

Targeted deletion of adipocytes by apoptosis leads to adipose tissue recruitment of alternatively activated M2 macrophages.

Obesity is frequently associated with an infiltration of macrophages into adipose tissue. Adipocyte dysfunction causes a phenotypic switch of macrophages from an alternatively activated M2-like phenotype towards a proinflammatory M1 phenotype. The cross talk between adipocytes and infiltrating immune cells, in particular macrophages, is thought to contribute to local and eventually systemic inflammation. Here, we tested the phenotypic impact of a lack of adipocytes on the inflammatory status of macrophages. We took advantage of the fat apoptosis through targeted activation of caspase-8 (FAT-ATTAC) mouse model that allows for the inducible system-wide elimination of adipocytes through a proapoptotic mechanism and followed the degree and type of inflammatory response upon ablation of live adipocytes. Analysis of depots 2 wk after elimination of adipocytes resulted in markedly reduced levels of adipose tissue and a robust down-regulation of circulating adipokines. Quantitative PCR and immunohistochemistry on epididymal and inguinal fat depots revealed an increase of the macrophage markers F4/80 and CD11c. Using polychromatic flow cytometry, we observed an up-regulation of alternatively activated M2 macrophage markers (CD206 and CD301) on the majority of F4/80 positive cells. Apoptosis of adipocytes is sufficient to initiate a large influx of macrophages into the remnant fat pads. However, these macrophages are alternatively activated, antiinflammatory M2 macrophages and not M1 cells. We conclude that adipocyte death is sufficient to initiate macrophage infiltration, and live adipocytes are required to initiate and/or sustain a proinflammatory response within the infiltrating macrophages in adipose tissue.