为了正常的体验网站,请在浏览器设置里面开启Javascript功能!
首页 > 脂肪形成

脂肪形成

2013-12-11 8页 pdf 2MB 17阅读

用户头像

is_405081

暂无简介

举报
脂肪形成 1338 VOLUME 19 | NUMBER 10 | OCTOBER 2013 nature medicine t e c H n i c a L r e P O r t s White adipose tissue displays high plasticity. We developed a system for the inducible, permanent labeling of mature adipocytes that we called the AdipoChaser mouse. We ...
脂肪形成
1338 VOLUME 19 | NUMBER 10 | OCTOBER 2013 nature medicine t e c H n i c a L r e P O r t s White adipose tissue displays high plasticity. We developed a system for the inducible, permanent labeling of mature adipocytes that we called the AdipoChaser mouse. We monitored adipogenesis during development, high-fat diet (HFD) feeding and cold exposure. During cold-induced ‘browning’ of subcutaneous fat, most ‘beige’ adipocytes stem from de novo–differentiated adipocytes. During HFD feeding, epididymal fat initiates adipogenesis after 4 weeks, whereas subcutaneous fat undergoes hypertrophy for a period of up to 12 weeks. Gonadal fat develops postnatally, whereas subcutaneous fat develops between embryonic days 14 and 18. Our results highlight the extensive differences in adipogenic potential in various fat depots. The ongoing obesity epidemic in both the western and developing worlds has raised awareness of the complex physiology of adipose tissue. It is widely appreciated that anatomically distinct adipose tis- sues differ substantially in their contributions to energy balance and nutrient homeostasis. Adipose tissue distribution is a strong predictor of the occurrence of the metabolic syndrome in the context of obesity. Obese individuals who preferentially expand visceral adipose tissue are at a greater risk for diabetes and cardiovascular disease than are equally obese individuals who store excess energy in subcutaneous adipose tissue1–3. In fact, the expansion of subcutaneous adipose tis- sue can be potently protective against metabolic complications of HFD feeding4,5. The mechanism by which individual adipose depots expand may also be a critical determinant of the metabolic syndrome in obesity. In principle, adipose tissue expansion can occur through an enlarge- ment in adipocyte size (hypertrophy) or an increase in adipocyte numbers (hyperplasia). Differentiated adipocytes are post-mitotic; therefore, hyperplasia represents an increase in de novo adipocyte formation (adipogenesis). Adipocyte hypertrophy is closely linked to adipose dysfunction: this pathological expansion of white adipose tis- sue (WAT) is a major component of the metabolic syndrome in obese individuals6. Both adipocyte hypertrophy and hyperplasia contrib- ute to adipose tissue expansion during HFD challenge6,7. However, it remains under debate whether adipogenesis is induced immedi- ately after HFD exposure and whether there are depot differences in response to HFD with respect to adipogenesis8,9. Recent studies have highlighted the critical role of in vivo adipogenesis as it relates to key adipogenic precursor cells10–15. WAT is known to have high physiological plasticity. Exposure to cold or pharmacological treatment with β-adrenergic receptor (β3) agonists that enhance lipolysis in adipocytes triggers the appearance of a subset of potentially beneficial adipocytes within the WAT that are positive for uncoupling protein 1 (UCP1) and share additional characteristics with brown adipocytes16–19. Unlike classic brown adipocytes, these ‘brown-like’ fat cells, termed beige adipocytes, do not derive from precursors that are positive for myogenic factor 5 (Myf5)20. Recent studies have suggested that in subcutaneous adi- pose tissue, newly induced brown adipocytes are not associated with precursor proliferation21 and may arise from the transdifferentiation of existing white adipocytes22–24. Conversely, others have found that there is a CD137+ precursor cell population that is at rest in subcuta- neous adipose tissue and can be activated to differentiate into brown adipocytes after appropriate stimulation15. However, to date, there has been no direct evidence found for the unambiguous lineage of these beige adipocytes. Leptin reporter mice have been valuable in identifying early waves of adipogenesis occurring developmentally25; however, inducible lineage-tracing mouse models to examine adipo- genesis are still lacking. To better understand the dynamics of adipocytes in different fat depots, we developed a doxycycline-inducible, mature adipocyte– specific tracing system that we refer to as the AdipoChaser mouse. We are thus in a unique position to answer the questions outlined above in vivo with a high degree of temporal resolution. By using a pulse-chase system that allows us to label all pre-existing mature adi- pocytes, we uncovered the differential adipogenic capacity of epidi- dymal and subcutaneous adipose tissue during HFD-induced adipose tissue expansion. Our studies also offer insights into the unresolved issue of whether brown-like fat cells induced by cold exposure or β3 agonist treatment in subcutaneous adipose tissue arise from existing mature adipocytes or derive from de novo differentiation. Most nota- bly, we found that cold exposure or β3 agonist stimulation induces massive white adipogenesis in epididymal adipose tissue. In addition, our labeling system allowed us to determine the exact developmental time frame of adipocyte differentiation in gonadal and subcutaneous adipose tissue. Tracking adipogenesis during white adipose tissue development, expansion and regeneration Qiong A Wang1, Caroline Tao1, Rana K Gupta1 & Philipp E Scherer1,2 1Touchstone Diabetes Center, Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, Texas, USA. 2Department of Cell Biology, The University of Texas Southwestern Medical Center, Dallas, Texas, USA. Correspondence should be addressed to P.E.S. (philipp.scherer@utsouthwestern.edu). Received 7 May; accepted 9 July; published online 1 September 2013; doi:10.1038/nm.3324 np g © 20 13 N at ur e A m er ic a, In c. A ll rig ht s re se rv ed . t e c H n i c a L r e P O r t s nature medicine VOLUME 19 | NUMBER 10 | OCTOBER 2013 1339 RESULTS The contribution of hyperplasia to adipose tissue expansion In order to study the fate of mature adipocytes under different meta- bolic challenges, we developed the AdipoChaser mouse, which is an inducible adipocyte-tagging system. This model is a combination of three transgenic lines that we and other labs have generated: the adi- ponectin promoter–driven tetracycline-on (Tet-on) transcription fac- tor rtTA (adiponectinP-rtTA)26, a Tet-responsive Cre (TRE-cre) line that can be activated by rtTA in the presence of doxycycline27 and a transgenic line carrying Rosa26 promoter–driven loxP-stop-loxP–β- galactosidase (Rosa26-loxP-stop-loxP-lacZ)28 (Fig. 1a). In the absence of doxycycline, there was no LacZ expression in mature adipocytes. After treatment with doxycycline, rtTA activates the TRE promoter to induce cre expression. Cre protein subsequently eliminates the floxed transcriptional stop cassette and permanently turns on LacZ expres- sion in all mature adipocytes present during doxycycline exposure. Cells can be stained blue with an appropriate β-galactosidase (β-gal) substrate. After 2 weeks of doxycycline treatment, all adipocytes in both the epididymal and subcutaneous white adipose tissues (eWAT and sWAT, respectively) of the triple transgenic (AdipoChaser) mice were uniformly labeled blue (Fig. 1b,c), reflecting the presence of β-gal activity. Other cell types, such as endothelial cells in the subcu- taneous adipose tissue, were not labeled (Fig. 1c). We also used β-gal substrate to stain adipocytes from control (containing only TRE-cre and Rosa26-loxP-stop-loxP-lacZ) mice treated with doxycycline and adipocytes from AdipoChaser mice kept on chow diet; none of these stainings showed a β-gal signal (Fig. 1b,c). We next tested the washout period of doxycycline in mice. We kept AdipoChaser mice on doxycy- cline diet for 7 d and then switched them to chow diet without doxy- cycline for time periods ranging from overnight to up to 3 d before analyzing them. Real-time quantitative PCR (qPCR) results showed that each group of AdipoChaser mice had a similar level of rtTA expression in subcutaneous adipose tissue (Supplementary Fig. 1). Cre expression levels in AdipoChaser mice after 3, 2 or 1 d or over- night doxycycline withdrawal were comparable to the expression levels in AdipoChaser mice kept on chow diet without doxycycline and were much lower than those in AdipoChaser mice treated con- tinuously in the presence of doxycycline (Supplementary Fig. 1). b eWAT Control with dox AdipoChaser without dox AdipoChaser with dox c sWAT Control with dox AdipoChaser without dox AdipoChaser with dox a adnP rtTA Doxycycline Rosa26 lacZcreTRE Rosa26 Stop lacZ loxP loxP Figure 1 Inducible labeling of mature adipocytes. (a) The inducible labeling system of mature adipocytes, produced by crossing adiponectinP- rtTA (adnP-rtTA) transgenic mice with TRE-cre and Rosa26-loxP-stop- loxP-lacZ transgenic mice. The triple transgenic mouse, called the AdipoChaser mouse, expresses rtTA in mature adipocytes but does not express LacZ in any cell type while maintained on food not containing doxycycline (dox). When doxycycline is included in the food, adipocytes that express rtTA will have the TRE promoter activated so that cre expression is induced. The Cre protein will specifically cut out the floxed transcriptional stop cassette and then turn on LacZ expression. Even after withdrawal of doxycycline from the food, these adipocytes will permanently express LacZ, whereas any new adipocytes that develop after doxycycline exposure will not express LacZ. (b,c) Representative β-gal (blue) staining of eWAT (b) and sWAT (c) in male control (mice with only TRE-cre and Rosa26-loxP-stop-loxP-lacZ) or AdipoChaser mice. Solid arrows (b,c), LacZ-positive cells; open arrows (b,c), LacZ-negative cells. Scale bar (black, shown in b, applies to b and c), 200 µm; (blue, shown in b, applies to the insets in b and c), 50 µm. Throughout the figure, n = 2 male mice per group. eWAT Chow 3 d Chow 59 da Chow 3 d + HFD 7 d Chow 3 d + HFD 35 d Chow 3 d + HFD 56 d Chow 3 d + HFD 89 d eWAT b sWAT Chow 3 d + HFD 7 d Chow 3 d + HFD 35 d Chow 3 d + HFD 56 d Chow 3 d + HFD 89 dd sWAT Chow 3 d Chow 59 dc Normal Hypertrophy Hypertrophy and hyperplasia Hypertrophy Prolonged HFD Prolonged HFD HFDeWAT Normal Hypertrophy HFDsWAT e Figure 2 HFD-induced adipose tissue hypertrophy and hyperplasia. (a–d) Representative β-gal staining of eWAT (a,b) and sWAT (c,d) from 9- to 10-week-old male AdipoChaser mice that were kept on doxycycline diet for 7 d followed by chow diet for 3 or 59 d (a,c) or chow diet for 3 d and HFD for 7, 35, 56 or 89 d (b,d). Solid arrows (b), LacZ-positive cells; open arrows (b), LacZ-negative cells. Scale bar (shown in a, applies to a–d), 200 µm. For a–d, n = 3 male mice per group. (e) Schematic model of the depot-dependent contribution of hyperplasia to adipose tissue expansion after HFD feeding. HFD-induced adipose tissue expansion is contributed mainly by hypertrophy in both eWAT and sWAT at the early stages. After prolonged HFD exposure (i.e., longer than 1 month), a wave of adipogenesis is preferentially initiated in eWAT (hyperplasia), but adipogenesis does not occur at measurable levels in sWAT. Adipocytes surrounded by blue circles represent old LacZ-positive cells, and adipocytes surrounded by white circles represent new LacZ-negative cells. np g © 20 13 N at ur e A m er ic a, In c. A ll rig ht s re se rv ed . t e c H n i c a L r e P O r t s 1340 VOLUME 19 | NUMBER 10 | OCTOBER 2013 nature medicine During the same time course, AdipoChaser mice had similar levels of LacZ expression throughout the doxycycline withdrawal periods that were much higher than those in mice never treated with doxycycline (Supplementary Fig. 1). These results show that an overnight with- drawal is sufficient to completely wash out doxycycline in mice. Using this system, we initially focused on the rate of appearance of de novo–differentiated adipocytes during HFD challenge. We first gave AdipoChaser mice doxycycline diet for 7 d to ensure uniform and permanent labeling of mature adipocytes with LacZ, which we followed with 3 d of chow diet to ensure that the doxycycline was fully washed out. Thereafter, we fed the mice either chow or HFD (60% of calories from fat) for various lengths of time. HFD feed- ing for 12 weeks increased the body weight of AdipoChaser mice by 51% (P < 0.001) as compared to the basal body weight of the mice before beginning the HFD, whereas AdipoChaser mice kept on chow had only a 15% increase in body weight (P < 0.001) (Supplementary Fig. 2a). The weights of epididymal adipose tissue and subcutaneous adipose tissue of 12-week HFD-fed AdipoChaser mice were increased by 84% (P < 0.001) and 163% (P < 0.001), respectively, compared to age-matched AdipoChaser mice kept on chow diet (Supplementary Fig. 2b,c). AdipoChaser mice fed with chow diet showed no new adipogenesis, as determined by the fact that all fat depots displayed nearly 100% positive β-gal staining in adipocytes, even at up to 59 d after doxycycline treatment (Fig. 2). Thus, extremely low levels of adipogenesis were present in both epididymal and subcutaneous adipose tissues (Fig. 2a,c). After HFD feeding for 7 d, adipocytes in both the epididymal and subcutaneous adipose tissues still showed nearly 100% LacZ labeling with no obvious morphological changes (Fig. 2b,d). When we kept mice on HFD for 35 d, the average size of the adipocytes in both depots increased markedly, reflecting a high capacity for cell hypertrophy (Fig. 2b,d). After 56 or 89 d of HFD feeding, epididymal adipose tissue showed a high adipogenesis rate, as determined by a large number of β-gal–negative cells (Fig. 2b); in contrast, the subcutaneous adipose depot maintained a relatively low rate of adipogenesis, as evidenced by nearly 100% LacZ labeling (Fig. 2d). These observations indicate that HFD-induced adipose tissue expansion is contributed mainly by hypertrophy during the first month of HFD. After prolonged HFD exposure (i.e., longer than 1 month), a wave of adipogenesis is preferentially initiated in epidi- dymal adipose tissue, whereas only negligible levels of adipogenesis occur in subcutaneous adipose tissue depots (Fig. 2e). These experi- ments reveal the surprising property of subcutaneous adipose tissue to use de novo adipogenesis only minimally as a mechanism to cope with chronic caloric excess. Beige adipocytes arise by de novo adipogenesis It is not clear whether the beige cells that are induced by cold exposure or β3 agonist treatment arise through transdifferentiation of exist- ing white adipocytes or by de novo adipogenesis from a subgroup of precursor cells. We therefore studied newly developed beige adi- pocytes within subcutaneous adipose tissue under those conditions. We pretreated AdipoChaser mice with doxycycline diet for 7 d to ensure uniform, permanent expression of LacZ in mature adipocytes, which we followed with 3 d of chow diet to wash out the doxycy- cline. Overnight cold exposure in doxycycline-pretreated mice on a chow diet showed clusters of cells within the subcutaneous adipose tissue that were smaller than normal white adipocytes and that dis- played negative β-gal staining (Fig. 3a). Three days of cold exposure induced massive browning of subcutaneous adipose tissue, with the AdipoChaser mice showing large areas of beige fat cells with multi- ple small lipid droplets, and most of these tissues had negative β-gal a b d e f g c CE overnight CE 3 d CE 3 d + RT 7 d CE 3 d with dox Beige Beige Beige Beige BeigeBeigeBeige Beige Beige White White White White White White White White WhiteWhite White White Merge Merge Merge Brightfield color Brightfield color Brightfield color DAPI DAPI DAPI Perilipin Ucp1 Cited1 RT 6 °C Mature white adipocyte A subgroup of precursors Beige adipocytes De n ovo differ entia tion Dedifferentiation Brightfield B&W Brightfield B&W Brightfield B&W β3 7 d Figure 3 Lineage of the brown-like adipocytes in subcutaneous adipose tissue after cold exposure. (a) Representative β-gal staining of sWAT from 10-week-old male AdipoChaser mice that were kept on doxycycline diet for 7 d followed by chow diet for 3 d and then exposed to cold (CE) overnight (left) or for 3 d (middle) or exposed to cold for 3 d followed by 7 d in room temperature (RT; right). (b) Representative β-gal staining of sWAT from 10-week-old male AdipoChaser mice that were on doxycycline diet before and during 3 d of cold exposure as a positive control group. (c) Representative β-gal staining of sWAT from 10-week-old male AdipoChaser mice on doxycycline diet for 7 d followed by chow diet for 3 d and then given 7 d of daily β3 agonist treatment. Solid arrows (a–c), LacZ-positive cells; open arrows (a,c), LacZ-negative cells. Scale bar (shown in a, applies to a–c), 100 µm. For a–c, n = 3 male mice per group. (d–f) Immunofluorescence staining for perilipin (green) (d), Ucp1 protein (green) (e) or Cited1 protein (green) (f) on slides prestained with β-gal. The male AdipoChaser mice in d–f were pretreated with doxycycline diet and exposed to cold for 3 d on a chow diet. Blue indicates pre-existing white adipocytes. Scale bar (shown in d, applies to d–f), 200 µm. The yellow dashed outlines indicate the borders between white and beige adipocytes. For d–f, n = 2 male mice per group. B&W, black and white. (g) Schematic model showing that the beige cell population arises predominantly from de novo adipogenesis rather than transdifferentiation. After cold exposure or β3 agonist treatment, most beige adipocytes are induced by differentiating from cell populations other than existing mature adipocytes (beige precursors) rather than through dedifferentiation of mature white adipocytes. np g © 20 13 N at ur e A m er ic a, In c. A ll rig ht s re se rv ed . t e c H n i c a L r e P O r t s nature medicine VOLUME 19 | NUMBER 10 | OCTOBER 2013 1341 staining (Fig. 3a). These new adipocytes were preserved for at least 7 d in the subcutaneous adipose tissue after we switched the mice back to room temperature (Fig. 3a). In mice that we kept on doxycycline diet throughout the cold exposure, ~100% of the beige adipocytes showed LacZ-positive signals, demonstrating that these newly emerging cells can indeed be labeled if the mouse remains exposed to doxycycline during cold exposure (Fig. 3b). We observed similar results in the β3 agonist–treated mice: most of the newly generated beige adipocytes in these mice were negative for β-gal staining (Fig. 3c). Immunofluorescence staining demonstrated that LacZ-negative cells that formed in the subcutaneous adipose tissue of AdipoChaser mice were positive for the lipid droplet–specific marker perilipin, Ucp1 and the beige cell marker Cbp/p300-interacting transactivator with Glu/Asp-rich C-terminal domain, 1 (Cited1)29, whereas the LacZ- positive blue adipocytes were positive for perilipin only (Fig. 3d–f). These results indicate that after cold exposure or β3 agonist treat- ment, most beige adipocytes are induced by de novo differentiation from cell populations other than existing mature adipocytes (i.e., Cited1+Cd137+ beige precursors) rather than by a transdifferentiating mature white adipocyte (Fig. 3g). Beige cells can also revert to a white adipocyte phenotype, as has been recently reported30 (Fig. 3g). Cold-induced adipogenesis in epididymal adipose tissue Previous studies have shown that the appearance of brown-like cells is observed mainly in subcutaneous adipose tissue. Very few of these newly emerging cells can be detected in the visceral fat20. Notably, after cold exposure during as short a time period as overnight, epididymal adipose tissue of mice pretreated with doxycycline on a chow diet showed num
/
本文档为【脂肪形成】,请使用软件OFFICE或WPS软件打开。作品中的文字与图均可以修改和编辑, 图片更改请在作品中右键图片并更换,文字修改请直接点击文字进行修改,也可以新增和删除文档中的内容。
[版权声明] 本站所有资料为用户分享产生,若发现您的权利被侵害,请联系客服邮件isharekefu@iask.cn,我们尽快处理。 本作品所展示的图片、画像、字体、音乐的版权可能需版权方额外授权,请谨慎使用。 网站提供的党政主题相关内容(国旗、国徽、党徽..)目的在于配合国家政策宣传,仅限个人学习分享使用,禁止用于任何广告和商用目的。

历史搜索

    清空历史搜索