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hypertrophy is closely linked to adipose dysfunction: this pathological expansion of white

adipose tissue (WAT) is a major component of the metabolic syndrome in obese

individuals 6. Both adipocyte hypertrophy and hyperplasia contribute to adipose tissue

expansion during HFD challenge 6,7. However, it remains under debate whether adipogenesis

is induced immediately after HFD exposure and whether there are depot differences in

response to HFD with respect to adipogenesis 8,9. Recent studies have highlighted the critical

role of in vivo adipogenesis as it relates to key adipogenic precursor cells 10–15.

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

adipocytes 16–19. 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 adipose tissue, newly induced brown

adipocytes are not associated with precursor proliferation 21 and may arise from the

transdifferentiation of existing white adipocytes 22–24

there is a CD137+ precursor cell population that is at rest in subcutaneous adipose tissue and

can be activated to differentiate into brown adipocytes after appropriate stimulation 15.

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 developmentally 25; however, inducible lineage-tracing mouse

models to examine adipogenesis 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 adipocytes, we uncovered the differential

adipogenic capacity of epididymal 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 notably, 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.Results

The contribution of hyperplasia to adipose tissue expansion

In order to study the fate of mature adipocytes under different metabolic 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

adiponectin promoter-driven tetracycline-on (Tet-on) transcription factor rtTA

(adiponectinP-rtTA )26, a Tet-responsive Cre (TRE-cre ) line that can be activated by rtTA in

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the presence of doxycycline 27 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 expression 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 subcutaneous 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 doxycycline diet for 7 d and then switched them to chow diet without doxycycline 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 overnight 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 continuously in the presence of doxycycline (Supplementary Fig. 1). 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 withdrawal 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 feeding 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

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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 epididymal adipose tissue, whereas only

negligible levels of adipogenesis occur in subcutaneous adipose tissue depots (Fig. 2e).

These experiments 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 existing white adipocytes or by de novo

adipogenesis from a subgroup of precursor cells. We therefore studied newly developed

beige adipocytes 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

doxycycline. 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 displayed 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 multiple small lipid droplets,

and most of these tissues had negative β-gal 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 treatment, 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 reported 30 (Fig. 3g).NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

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 fat 20. 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

numerous β-gal–negative cells (Fig. 4a and Supplementary Fig. 3). Unlike beige adipocytes,

these new cells were larger in size and had only a single large lipid droplet. Three days of

cold exposure further increased the number of β-gal–negative cells in epididymal adipose

tissue (Fig. 4a), and the newly generated adipocytes remained present even after we

switched the mice back to room temperature (Fig. 4a). As a control, we used mice kept on a

doxycycline diet throughout cold exposure and found that every adipocyte in these mice

showed a LacZ-positive signal, demonstrating that these newly emerging cells in epididymal

adipose tissue can be labeled if the mouse remains exposed to doxycycline during cold

exposure (Fig. 4b). Similarly, in the β3 agonist–treated mice, β-gal–negative white

adipocytes appeared in epididymal adipose tissue (Fig. 4c). In order to provide further

evidence that adipogenesis in the epididymal adipose tissue is triggered after cold exposure,

we isolated RNA from the epididymal adipose tissue of mice that we either left unexposed

or exposed to cold for 3 d for qPCR analysis of lacZ expression and other adipocyte

markers. Compared to mice kept at room temperature, mice that were exposed to cold had

decreased lacZ expression in epididymal adipose tissue (by 53%, P < 0.05), with no change

in rtTA expression or that of other adipocytes markers, such as Adipoq (adiponectin) or

Pparg2 (peroxisome proliferator-activated receptor γ2) (Fig. 4d). Immunofluorescence

staining showed that these β-gal–negative cells were perilipin positive but Ucp1 negative

(Fig. 4e,f). These cells were therefore live cells that had a gene expression pattern consistent

with that of classic white adipocytes.

We next wanted to determine whether cold exposure increases the tissue mass of epididymal

adipose tissue. Three days of cold exposure slightly but significantly decreased the body

weight of wild-type mice (by 6%, P < 0.05) (Supplementary Fig. 4a). The weights of both

epididymal and subcutaneous adipose tissues were decreased after cold exposure (by 32%, P

< 0.05) and 36% (P < 0.001), respectively) compared to mice kept at room temperature

(Supplementary Fig. 4b). This observation indicates that despite ongoing adipogenesis in

epididymal adipose tissue during cold exposure, epididymal adipose tissue mass does not

increase overall but instead decreases. Taken together, our data provide strong evidence that

widespread browning occurs in subcutaneous adipose tissue through de novo adipogenesis

during cold exposure or β3 agonist stimulation. In parallel, there is a substantial amount of

de novo adipogenesis of white adipocytes that occurs under these conditions, primarily in

epididymal adipose tissue (Fig. 4g).

Adipogenesis during development

Having examined the behavior of fat pads under different conditions in adult mice, we then

studied commitment to the adipocyte lineage during development. In order to label

adipocytes during embryogenesis, we exposed AdipoChaser mice to doxycycline diet for

various time periods during pregnancy and analyzed the offspring at postnatal day (P) 28 or

56 (Fig. 5). In gonadal adipose tissue, both male and female mice whose mothers were on

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doxycycline during embryonic day (E) 9–16 or E19–P4 showed very few LacZ-positive cells once the mice were 28 days old (Fig. 5a). In AdipoChaser mice whose mothers were on doxycycline until P10, most of the adipocytes in the gonadal adipose tissue were LacZ

negative on P28 (Fig. 5a and Supplementary Fig. 5) or P56 (Fig. 5c). For mice whose

mothers were on doxycycline during the period E19–P20, large areas of the adipocytes in the gonadal adipose tissue were LacZ positive when the mice were 28 days old, but there

were also numerous adipocytes that developed after P20, as judged by negative LacZ

staining (Fig. 5a). These data show that gonadal adipocytes undergo differentiation postnatally for the most part, and this differentiation happens gradually over a relatively

long period of time after birth.

In contrast, subcutaneous adipose tissue showed a rather distinct pattern. None of the male

AdipoChaser mice whose mothers were on doxycycline during E7–E14 showed any LacZ-

positive adipocytes when examined on day 28, whereas female AdipoChaser mice under the

same conditions showed a few (<5%) LacZ-positive adipocytes (Fig. 5b). However,

adipocytes in subcutaneous tissue of mice whose mothers were on doxycycline during E9–

E16 showed a heterogeneous pattern of LacZ-positive cells, with some regions carrying

more than 90%, and other regions displaying less than 50%, LacZ-positive cells (Fig. 5b).

AdipoChaser mice whose mothers were on doxycycline treatment during E9–E18 or E19–

P10 showed nearly 100% LacZ-positive cells in subcutaneous adipose tissue (Fig. 5b and

Supplementary Fig. 5). These observations indicate that for subcutaneous adipocytes,

adipocyte commitment and differentiation are initiated during E14–E18 in both genders.

Large areas of the subcutaneous adipose tissue were still very small and lacked mature

adipocyte morphology in the perinatal phase (28 d) (Fig. 5b). When mice whose mothers

were on doxycycline during E19–P10 were 56 days old, adipocytes in the subcutaneous

adipose tissue in both genders remained LacZ positive and were fully differentiated with

relatively even cell sizes and single lipid droplets (Fig. 5d). Notably, there were also some

LacZ-positive nonadipocytes around the vascular structure in the subcutaneous adipose

tissue (Fig. 5b), even in mice whose mothers were on doxycycline during E7–E14 (Fig. 5b).

These results suggest that, unlike gonadal adipocytes, subcutaneous adipocytes are

committed and differentiated much earlier during embryogenesis. Although subcutaneous

adipose tissue takes more time for complete differentiation, the number of adipocytes

remains very stable postnatally (Fig. 5e).Discussion

Assessments of in vivo adipogenesis have relied on measurements of cells size or BrdU

incorporation into adipocytes 8,9. The underlying assumption in using such methods is that

adipocyte size reflects the age of the adipocyte and that preadipocyte proliferation precedes

adipogenesis. Here we applied a genetic model that permitted the uniform labeling of mature

adipocytes in WAT. By using this model, we found that the rate of white adipogenesis in

both epididymal and subcutaneous adipose tissue in adult mice was very low when the

animals were maintained on a chow diet. This is consistent with human studies that

demonstrated previously that only about 10% of adipocytes are renewed annually in

adults 31. During the early stages of HFD exposure (i.e., within about 1 month of beginning

the diet), we observed that hypertrophy was the main phenomenon contributing to adipose

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tissue expansion in both epididymal and subcutaneous adipose tissues. At more prolonged stages of HFD exposure (i.e., 2 months), a high capacity for adipogenesis was apparent in gonadal adipose tissues. In contrast, subcutaneous adipose tissues maintained an extremely low rate of adipogenesis, even after 2 months of HFD exposure. These results highlight that the adipogenesis rates of different fat pads differ extensively. This helps to better explain phenomena related to prolonged HFD exposure. It also underscores the fact that phenomena related to the isolation of stromal vascular fractions (SVFs) from various fat pads and their ability to in vitro differentiate do not relate to their in vivo capacity to undergo adipogenesis.In fact, cultures of stromal vascular cells derived from subcutaneous fat regions readily give rise to adipocytes in vitro ; however, subcutaneous adipogenesis in vivo seems to be limited,at least in the male mice that we analyzed here.Brown adipocytes have the potential to be metabolically beneficial, especially for obese individuals, as they have the potential for enhanced energy expenditure after proper stimulation. Although we cannot exclude that there is some degree of transdifferentiation,our findings demonstrate that most of the newly emerging beige adipocytes that appear in subcutaneous depots are not derived from existing white adipocytes. They therefore must derive from a precursor cell type that undergoes differentiation rather than from the transdifferentiation of existing adipocytes. This is consistent with a recent report that demonstrated that brown-like adipocytes derive from Cd137+ cells 15. BrdU incorporation into cells has been widely used as an indicator of adipogenesis. However, the lack of BrdU incorporation only highlights that there is no cell-proliferative step in subcutaneous adipose tissue 21. If beige adipocytes derive from resting precursor cells 15 with very limited proliferation, the newly formed cells will also be BrdU negative.

Our experiments reveal another interesting phenomenon that is difficult to detect in the

absence of the type of labeling techniques that we used here. We found that cold exposure or β3 agonist stimulation induced widespread white adipocyte differentiation in the epididymal adipose tissue, a phenomenon that is markedly different from the browning effect seen in subcutaneous adipose tissues under these conditions. The current consensus in the field views epididymal adipose tissue as relatively nonresponsive to adrenergic stimulation, and our data support this view when we compare the potential for browning of the epididymal depot with that of the subcutaneous depots. However, our LacZ pulse and chase experiments revealed that epididymal adipose tissue is potently responsive to cold stimuli. This is an interesting phenomenon, and one previous report has suggested that this may be the https://www.wendangku.net/doc/c516850779.html,ler and Faust reported in 1982 that maintaining rats for 24 weeks in the cold was associated with an increase in adipocyte numbers selectively in the epididymal adipose tissue, a phenomenon they did not observe in any other fat pads 32. They contrast this

development to HFD-induced changes that induce proliferative events in retroperitoneal but not epididymal adipose tissue. Our findings suggest that this cold-induced adipogenesis in epididymal adipose tissue is a very fast process, occurring within a few days of cold exposure.

In recent years, a number of studies have focused on the characteristics of fat progenitor cells. Mesenchymal stem cells isolated from adipose tissue have been shown to have the potential to differentiate into various cell types, including adipocytes 33. It is known that

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adipocyte precursor stem cells are present in the pool of stromal vascular cells (the SVF) of adipose tissue. These cells can be differentiated into mature adipocytes both in vitro and in vivo 11. Similarly to Ppar-γ, zinc finger protein 423 (Zfp423) has been discovered to be a marker of committed preadipocytes 12,13. Expression analyses of PPAR-γ or Zfp423 have revealed that adipogenic precursors are located around the vascular wall of adipose tissue 10,13. However, it is still unknown whether the progenitor cells that give rise to white adipocytes during the normal development of new adipose tissue share the same characteristics and respond to the same stimuli as their counterparts that undergo adipose tissue adipogenesis and regeneration during adulthood and aging. It is also not understood how many features there are in common between visceral and subcutaneous precursors. It has been shown that some pad-specific characteristics are preserved after isolation and in vitro differentiation of these cells 34. Our lineagetracing studies showed that epididymal adipose tissue has a relatively high potential to undergo adipogenesis under various environmental challenges, such as HFD challenge or cold exposure. In addition, the time frame of gonadal adipose tissue development is completely different from that of subcutaneous adipose tissue. It is therefore entirely possible that these two depots may not share the same progenitors. Alternatively, precursors from these two depots either do not respond to the same stimuli to differentiate or are differentially exposed to those signals because of differential spatial organization. It is noteworthy that the SVF from subcutaneous adipose tissues differentiates more robustly and readily in vitro than the SVF from epididymal adipose tissue 35–37. This has led many to believe that this observation may underlie the physiological basis of why subcutaneous WAT in humans seems to expand predominantly through hyperplasia 35–37. However, the data here clearly indicate that in mice, epididymal adipose tissue is more prone to undergoing differentiation than is subcutaneous adipose tissue. Understanding the underlying mechanisms for these phenomena is more than an academic exercise. The location of adipose tissue accumulation is of paramount importance with respect to clinical readouts for the metabolic syndrome in the clinic 38,39. We have published several preclinical models that highlight the protective effects that are associated with subcutaneous adipose tissue expansion 4,5. The in vitro studies highlight the potential for subcutaneous adipogenesis. However, under most conditions, subcutaneous adipogenesis is not activated in vivo . This highlights the existence of a critical ‘switch’ that must be activated in vivo to steer local events away from the hypertrophic growth of existing adipocytes and initiate the more beneficial cascade that leads to increases in the number of adipocytes.In summary, by using an inducible mature adipocyte lineage–tracing system, the AdipoChaser mouse, we characterized the differentiation of adipocytes in various adipose tissues during development and explored the dynamics of these adipose tissues under various physiological challenges. The variation of timing and rates of adipogenesis in different adipose tissues highlight the unique characteristics of the depots that should each be treated as a separate ‘miniorgan’. The hope is that we will develop approaches in the future that allow us to manipulate these fat pads in a depot-specific fashion.

Methods Methods and any associated references are available in the online version of the paper.

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Note: Any Supplementary Information and Source Data files are available in the online version of the paper.

Online Methods Mice Mice were maintained on a 12-h dark, 12-h light cycle and were housed in groups of three to five with unlimited access to water and food (chow: number 5058, lab diet; HFD: number D1292, Research diet; doxycycline diet (600 mg per kg diet): S4107, Bio-Serv, as indicated in the individual experiments). The Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center, Dallas, approved all animal experiments.All mice were on the C57BL/6J background. The adnP-rtTA mouse was generated as previously described 26. TRE-cre and Rosa26-loxP -stop-loxP-lacZ lines were obtained from Jackson Laboratories. For lineage-tracing studies, adnP-rtTA mice were intercrossed to the TRE-cre and Rosa26-loxP -stop-loxP-lacZ strains, and we refer to these triple transgenic mice as AdipoChaser mice.β-gal staining of adipose tissue Mice at 10–12 weeks of age were anesthetized and perfused with 0.2% glutaraldehyde in PBS. Fat depots of interest were removed into a 6-cm cell culture dish containing 0.2%glutaraldehyde and cut into small slices. The sliced tissue was washed with rinse buffer (100mM sodium phosphate, 2 mM MgCl 2, 0.01% sodium deoxycholate and 0.02% NP-40) three times and then soaked in β-gal staining buffer (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide and 1 mg ml -1 β-gal substrate in rinse buffer) for 48 h at room

temperature with mild shaking. After developing the β-gal stain, the sliced tissue was moved into 10% formalin overnight for post-fixation. The post-fixed tissue was processed with a standard paraffin tissue-embedding protocol to produce sections. After paraffin embedding and sectioning, tissues were counterstained with nuclear fast red. The staining was imaged using a Coolscope microscope (Nikon). Each β-gal stain image is a representative image from n = 2–3 mice out of two to three independent experiments.

Cold exposure and β3 agonist treatment

For cold exposure experiments, 10- to 11-week-old male AdipoChaser mice were kept in individual cages and maintained on chow diet or doxycycline diet for 3 d before cold exposure. During cold exposure, mice were kept on chow diet or doxycycline diet in

individual cages and housed in a cold cabinet (6 °C) for the indicated times with free access to food and water. Alternatively, 10-week-old mice were treated with β3 agonist

(CL-316243, Sigma) at 1 mg per kg body weight daily by intraperitoneal injections for 7 d while on a chow diet.

Immunofluorescence staining

Adipose tissues were prestained with β-gal solution and fixed in 10% PBS-buffered formalin overnight. Formalin-fixed, paraffin-embedded sections were blocked in PBS plus Tween-20(PBST) with 5% BSA. The primary antibodies used were to Ucp1 (ab23841, Abcam, 1:200),perilipin (1:200) (a kind gift of A. Greenberg, Tufts University) and Cited1 (ab15096,

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Abcam, 1:300); the secondary antibody used was Alexa Fluor 488 goat antibody to rabbit IgG (H+L) (A-11008, Invitrogen, 1:200)). Slides were counterstained with DAPI. Images were acquired using an AxioObserver epifluorescence microscope (Zeiss).

Quantitative real-time RT-PCR

Total RNA from mouse tissues was isolated using the RNeasy Mini Kit (QIAGEN). First-strand cDNA was synthesized with M-MLV reverse transcriptase and random hexamer primers (Invitrogen) from 1 μg of RNA. Real-time quantitative PCR was done with the SYBR Green PCR system (Applied Biosystems) using Gapdh as an internal control for normalization. The primers used were as follows (given as forward and reverse,

respectively): rtTA (5′-AGTCATTCCGCTGTGCTCTC-3′, 5′-

GCTCCTGTTCCTCCAATACG-3′), lacZ (5′-ACTATCCCGACCGCCT TACT-3′, 5′-GTGGGCCATAATTCAATTCG-3′), Adipoq (5′-GTTGCAA GCTCTCCTGTTCC-3′, 5′-ATCCAACCTGCACAAGTTCC-3′), Pparg2 (5′-TGCACTGCCTATGAGCACTT-3′, 5′-AACCATTGGGTCAGCTCT TG-3′), Gapdh (5′-AACTTTGGCATTGTGGAAGG-3′, 5′-ACACATTG GGGGTAGGAACA-3′).

Statistical analyses

Data are presented as the means ± s.e.m. Differences were analyzed by unpaired two-tailed t test between two groups. There was no randomization or blinding during data analysis. No statistical method was used to predetermine sample size.Supplementary Material Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

We thank the University of Texas Southwestern Histology Core for assistance in embedding and processing tissue samples, especially J.M. Shelton for suggestions and discussions on the protocol of LacZ staining of adipose tissues. We also thank A.S. Greenberg from Tufts University for providing perilipin antibody. This study was supported by US National Institutes of Health (NIH) grants R01-DK55758, P01-DK088761 and R01-DK099110(P.E.S.). Q.A.W. is supported by a postdoctoral fellowship from the American Diabetes Association (7-11-MN-47).R.K.G. is supported by NIH grants K01-DK090120-02 and R03-DK099428 and the Searle Scholars Program (Chicago, Illinois).

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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.

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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.

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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

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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 NIH-PA Author Manuscript

existing mature adipocytes (beige precursors) rather than through dedifferentiation of mature

white adipocytes.

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Figure 4.

Adipogenesis in epididymal adipose tissue during cold exposure. (a ) Representative β-gal staining of eWAT from AdipoChaser mice after overnight (left) or 3 d of cold exposure (middle) or 3 d of cold exposure followed by 7 d in room temperature (right). (b )

Representative β-gal staining of eWAT from 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 eWAT from AdipoChaser mice after 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 )

Relative mRNA expression levels of rtTA, lacZ, Adipoq and Pparg2 in eWAT from mice at room temperature or exposed to cold for 3 d. Gapdh was used as an endogenous control. n =11 male mice per group. Data are shown as the mean ± s.e.m. *P = 0.003 by unpaired two-tailed t test compared to the room temperature group. (e,f ) Immunofluorescence staining for perilipin (green) (e ) and Ucp1 protein (green) (f ) on slides prestained with β-gal. The male AdipoChaser mice in e and f were pretreated with doxycycline diet and exposed to cold for 3 d on a chow diet. Scale bar (shown in e , applies to e and f ), 200 μm. (g ) Schematic model of de novo adipogenesis of white adipocytes in eWAT during cold exposure or β3 agonist

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treatment. Unlike sWAT, eWAT responds to cold or β3 agonist treatment by initiating

massive adipogenesis. Adipocytes surrounded by blue circles represent old LacZ-positive

cells, adipocytes surrounded by white circles represent new LacZ-negative cells, and NIH-PA Author Manuscript

adipocytes surrounded by dashed blue circles represent potential adipocyte death. NIH-PA Author Manuscript

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Figure 5.

Development of epididymal and subcutaneous adipocytes during embryonic and postnatal development. (a-d ) Results from AdipoChaser mice that were on doxycycline diet for the indicated number of days during embryonic and postnatal development that were thereafter kept on chow diet. (a ) Representative β-gal staining of gonadal WAT (gWAT) from 28-day-old female mice (top) and male littermates (bottom). The mothers of these mice were on doxycycline diet during E9-E16, E19-P4, E19-P10 or E19-P20, as indicated. (b )

Representative β-gal staining of sWAT from 28 day-old-female (top) and male (bottom)mice. The mothers of these mice were on doxycycline diet during E7-E14, E9-E16, E9-E18or E19-P10, as indicated. (c,d ) Representative (3-gal staining of gWAT of 56-day-old

female (c , top) and male (c , bottom) mice and of sWAT from 56-day-old female (d , top) and male (d , bottom) mice. The mothers of these mice were on doxycycline diet during E19-P10. Solid arrows (a–c ), LacZ-positive cells; open arrows (a–c ), LacZ-negative cells. Scale bar (black, shown in a , applies to a–d ). 200 μm; (blue, shown in b , applies to the insets in b ), 50 μm. For a–d , n = 2 mice per group. (e ) Schematic model of the development of

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gWAT and sWAT. Adipocytes in the gWAT are differentiated postnatally between birth and

sexual maturation, whereas all the adipocytes in the subcutaneous adipose tissue start to

differentiate between E14 and E18, but the differentiation takes much longer and finishes NIH-PA Author Manuscript

postnatally. Yellow adipocytes represent adipocytes differentiated before birth, and green

adipocytes represent adipocytes differentiated postnatally. NIH-PA Author Manuscript

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