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Cell Reports, Volume 2, Issue 4, 976-990, 11 October 2012
Consecutive Positive Feedback Loops Create a Bistable Switch that Controls Preadipocyte-to-Adipocyte Conversion
Byung Ouk Park1, Robert Ahrends1 and Mary N. Teruel1, ,
1 Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA
Graphical Abstract
► A quantitative molecular model of adipogenesis is developed ► Multiple, consecutive feedback loops from PPARγ drive adipogenesis ► The circuit design with sequential, delayed loops prevents accidental triggering ► Insulin-regulated fat accumulation is graded and occurs only after the switch is made
- Adipogenesis, or the conversion of proliferating preadipocytes into nondividing adipocytes, is an important part of the vertebrate weight-maintenance program. It is not yet understood how and when an irreversible transition occurs into a distinct state capable of accumulating lipid. Here, we use single-cell fluorescence imaging to show that an all-or-none switch is induced before lipid accumulation occurs. Conversion begins by glucocorticoid and cAMP signals raising C/EBPβ levels above a critical threshold, triggering three consecutive positive feedback loops: from PPARγ to C/EBPα, then to C/EBPβ, and last to the insulin receptor. Experiments and modeling show that these feedbacks create a robust, irreversible transition to a terminally differentiated state by rejecting short- and low-amplitude stimuli. After the differentiation switch is triggered, insulin controls fat accumulation in a graded fashion. Altogether, our study introduces a regulatory motif that locks cells in a differentiated state by engaging a sequence of positive feedback loops.
Adipocytes, or fat cells, are essential for human health, carrying out critical functions including cushioning and insulating the body and internal organs, storing up to 80%–90% of the body's energy, and regulating glucose homeostasis and energy metabolism by secreting key hormones such as leptin, adiponectin, and TNF-α (Ahima and Flier, 2000; Rosen and Spiegelman, 2006). With the current epidemic of obesity and the strong correlations of obesity with diabetes, cardiovascular disease, and cancer, understanding the molecular mechanisms underlying adipogenesis, or the conversion of dividing preadipocytes into nondividing, lipid-accumulating fat cells, is of great scientific and medical interest. Many regulatory factors have been implicated in adipogenesis and have been depicted in summary diagrams (i.e., Cristancho and Lazar, 2011; Farmer, 2006; Lowe et al., 2011). However, static diagrams are inadequate for understanding the key steps in a dynamic and complex process like adipogenesis. In addition, to understand if, when, and how a clear commitment decision is made and a cell transitions irreversibly from a distinct preadipocyte state into a distinct adipocyte state requires measurements at the single-cell level. Such a single-cell analysis has not yet been performed during adipogenesis.
Adipogenesis occurs over several days and can be triggered by a number of hormonal stimuli. Several cell models have been established to study adipogenesis in vitro (Green and Kehinde, 1976; Wolins et al., 2006). In these models, adipogenesis is induced by the addition of glucocorticoid and insulin together with different strategies to increase cAMP. Key players in the transcriptional network controlling adipogenesis include the transcription factors C/EBPβ and C/EBPα and the nuclear receptor PPARγ, which is often described as a master regulator because it has been shown to be both necessary and sufficient for fat cell differentiation (Tontonoz and Spiegelman, 2008). Expression of C/EBPβ has been shown to induce the expression of PPARγ (Wu et al., 1996), most likely due to direct regulation because C/EBP binding sites have been identified in the PPARγ promoter (Zhu et al., 1995; Fajas et al., 1997). Previous work showed that a positive feedback exists between PPARγ and C/EBPα, and it has been suggested that this positive feedback is important to induce a terminal differentiated state (El-Jack et al., 1999; Rosen et al., 2002; Wu et al., 1999). However, positive feedbacks in cell regulatory systems are common, and their function in many cases is simply to amplify a transmitted signal (Brandman and Meyer, 2008). To trigger an irreversible decision or bistable switch, a cooperative regulatory step is required in addition to positive feedback. Also, in most known biological switch mechanisms such as the oscillations in Ca2+ signaling and the G2/M cell-cycle decision, cells rely on more than one positive feedback (Brandman et al., 2005). Neither multiple positive feedbacks nor cooperativity in the activation steps has been described in adipocyte differentiation.
Even the presence of multiple, cooperative positive feedbacks does not prove that a bistable, irreversible, or differentiated state is induced; this depends further on the specific enzymatic parameters. To show that a bimodal state is induced, single-cell experimental data using markers for the feedback regulators are first needed. To then prove that such a bimodal state is irreversible or bistable, one has to show that the inducing signals can be lowered or removed without losing the new differentiated state created by these feedback regulators (Pomerening et al., 2003; Yao et al., 2008). Finally, it has not yet been determined whether the induction of a bistable differentiation switch occurs independently of fat accumulation because current protocols typically use lipid accumulation as the marker for the differentiated state. These considerations provided the incentive for the studies we pursued here to uncover the molecular mechanisms triggering a potential irreversible bistable switch, and also to determine whether the triggering of such a switch precedes, coincides, or involves lipid synthesis.
To achieve this goal, we developed and applied an image-based approach to simultaneously quantify multiple key parameters in thousands of single cells over the time course of adipogenesis. Our analysis showed that the fat cell differentiation process is bimodal and that a clear decision is made early in differentiation before lipogenesis occurs. We identified a reinforcing feedback loop from PPARγ back to C/EBPβ that engages with a marked delay after a first positive feedback between PPARγ and C/EBPα. This delay is caused by a requirement for higher PPARγ activity for the second feedback to be triggered. We then identified a third commitment step, in which PPARγ expression is further boosted by a positive feedback between PPARγ and the insulin receptor that again engages with a delay after the first two positive feedbacks. We used these single-cell measurements to generate a quantitative model of the differentiation decision. Together with experimental data, model analysis showed that this consecutive feedback loop design is uniquely suited to lock cells in a differentiated state. Thus, our study introduces a regulatory design whereby multiple positive feedback loops sequentially engage with time delays to generate a robust transition to a terminally differentiated state.
Single-Cell Analysis of Adipogenic Transcription Factor Expression and Lipid Droplet Formation
To identify a potential bistable switch in the adipocyte differentiation path, we developed a multiparameter, single-cell assay to measure expression of key transcription factors and lipid droplet content over the time course of adipogenesis in both 3T3-L1 cells, a mouse embryo-derived cultured adipocyte model (Green and Kehinde, 1976), as well as OP9 cells, a bone marrow-derived adipocyte model. We and others have verified that OP9 and 3T3-L1 cells have similar adipocyte differentiation characteristics, although OP9 cells do differentiate faster than 3T3-L1 cells (Figures S1A and S1B; Wolins et al., 2006). OP9 cells represent late-stage preadipocytes and thus are more advanced in the differentiation process to become adipocytes (Wolins et al., 2006).
Figure 1A shows a schematic representation of transcriptional regulators that have been shown to control adipogenesis. C/EBPβ expression is upregulated by glucocorticoid and cAMP (Yeh et al., 1995), and PPARγ expression by C/EBPβ, C/EBPα, and insulin (Kim et al., 1998), with PPARγ then driving adipogenesis. Adipogenesis is commonly induced by growing preadipocyte cells such as OP9 or 3T3-L1 cells to confluency and then applying an adipogenic mixture consisting of insulin, fetal bovine serum (FBS), dexamethasone (dex), which is a synthetic glucocorticoid, and 3-isobutyl-1-methylxanthine (IBMX), which is an inhibitor of phosphodiesterase that increases cAMP levels. After 2 days, the glucocorticoid and cAMP stimuli are removed, and the media are replaced with media containing only insulin and FBS.
We performed single-cell image analysis of adipogenesis in both OP9 and 3T3-L1 cells (Figures 1B and S1A). Cells were plated in 96-well plates, induced to differentiate by the addition of insulin, glucocorticoid, and cAMP stimuli, fixed at different time points after induction, and stained with antibodies to quantify the expression level of the key adipogenic transcription factors. When we averaged the resulting antibody intensities from cells fixed at each day of adipogenesis, we observed a sequential order of events, similar to previously published western blot results by Farmer, 2006. In both OP9 cells and 3T3-L1 cells, maximal average C/EBPβ expression occurred 1–2 days after the induction of adipogenesis, preceding the induction of maximal PPARγ and C/EBPα expression, and followed after a delay by maximal lipid droplet formation (Figures 1C and S1B).
Bimodal Induction of PPARγ, C/EBPα, and C/EBPβ
To further explore the relationship between the transcription factors, we carried out single-cell, multiparameter analysis (Figure S1C). In contrast to analysis of population averages, histograms that plotted the concentration of the transcription factors in each of approximately 25,000 single cells showed bimodal expression of PPARγ, C/EBPα, and C/EBPβ, starting at day 3 of adipogenesis even though the cells had been uniformly stimulated (Figure 1D). Just as separating proteins out on a two-dimensional gel results in better resolution than separating in a one-dimensional gel, using two parameters to plot the histograms better resolved the bimodal nature of the transcription factor induction. Each of the panels in Figure 1E shows a dual-parameter histogram analysis that plotted the frequency at each day of differentiation at which individual cells had a given concentration of PPARγ and C/EBPβ. At day 0, all the cells had low PPARγ and low C/EBPβ. At days 1 and 2, the cells had slightly higher PPARγ and significantly higher C/EBPβ than at day 0. At day 3, two populations of cells were clearly evident, indicating that sometime between days 2 and 3, a subpopulation of cells reverted back into a low PPARγ and low C/EBPβ state, whereas a second kept increasing their high PPARγ and high C/EBPβ level. Strikingly, this transition into the high PPARγ and high C/EBPβ state occurred early in adipogenesis, 1 day before accumulation of lipid, which is the usual marker of terminal differentiation.
Our population-averaged results shown in Figure 1C initially suggested that for adipogenesis to occur, C/EBPβ expression first drops at day 2 to about half before PPARγ and C/EBPα reach maximal expression (Yeh et al., 1995). However, single-cell analysis led to a different conclusion (Figure 1E, schematics). After removal of the glucocorticoid and cAMP stimuli, the expression of both C/EBPβ and PPARγ further increased in a fraction of cells, whereas in the remaining cells, the expression of both C/EBPβ and PPARγ dropped back to basal in both OP9 (Figure 1E) and 3T3-L1 cells (Figure S1D). Thus, even though all cells experienced the same differentiation-inducing stimulus, this remarkable switch behavior resulted in two groups of cells with distinct levels of PPARγ and C/EBPβ and with increasing amounts of differentiation inducers resulting in increasing numbers of cells in the high PPARγ-high C/EBPβ differentiated cell group (Figure S1E). This bifurcation suggested that each cell undergoes an all-or-none cell fate decision to either commit to differentiation or to revert to the low PPARγ-low C/EBPβ preadipocyte state.
PPARγ Is Regulated by Two Positive Feedbacks to C/EBPα and to C/EBPβ
To enable the existence of two stable states, a system typically requires positive feedback, as well as one or more cooperative regulatory steps. Figure 2A shows a schematic representation of how positive feedback can help create such a decision. In the simplest case of positive feedback, the change in x is linearly related to the amount of y and vice versa. At steady-state conditions, the curves representing the dependence of x on y (blue line) and y on x (red line) will intersect at two points: one stable, and one unstable (Figure 2A, middle panel). If y is instead cooperatively related to x, for example if y is a gene that is only transcribed when three binding sites for transcription factor X in its promoter are occupied, this cooperative relationship can be described by a Hill equation and plotted as a sigmoidal curve (Figure 2A, right panel, red line). With this added cooperativity the curves representing the dependence of x on y (blue line) and y on x (red line) will now intersect at three points at steady-state conditions: two stable states and one unstable state. Positive feedback ensures that the system cannot rest in intermediate states, and cooperativity filters small signals out, allowing the system to have a stable off as well as a stable on-state (Ferrell and Xiong, 2001).
We first confirmed that a previously described positive feedback loop exists between PPARγ and C/EBPα (Rosen et al., 2002; Wu et al., 1999) by siRNA-mediated knockdown of PPARγ and C/EBPα expression, which reduced the expression of C/EBPα and PPARγ, respectively (Figure 2B, left and middle panels). However, one feedback loop—unless highly cooperative—is not enough to generate a bistable switch (Brandman and Meyer, 2008). We therefore searched for other potential feedback loops that could contribute to the bimodal patterns observed in Figure 1. As expected because C/EBPβ has been shown to act upstream of PPARγ (Yeh et al., 1995), knockdown of C/EBPβ reduced PPARγ expression (Figure 2B, left panel). However, when we used siRNA to suppress PPARγ expression, we found, especially at days 3 and 4, that C/EBPβ expression was also markedly reduced (Figure 2B, right panel), arguing that PPARγ was able to regulate the expression of its upstream activator C/EBPβ and suggesting that a second positive feedback links PPARγ and C/EBPβ. The existence of such a feedback loop is also supported by promoter binding studies that showed interactions of C/EBPβ with the PPARγ promoter (Schmidt et al., 2011) and of PPARγ with the C/EBPβ promoter (Mikkelsen et al., 2010).
Our siRNA data showed that this feedback loop between PPARγ and C/EBPβ engaged only 3–4 days after induction of adipogenesis, providing an important second boost to C/EBPβ expression. This suggested that a main role of this feedback is to keep C/EBPβ levels high after 48 hr independently of the initial glucocorticoid receptor (GR) and cAMP stimulation. This positive feedback between PPARγ and C/EBPβ therefore has the characteristics of a stabilizing switch mechanism that keeps PPARγ and C/EBPβ autonomously high even after the initiating stimulus is removed.
To confirm the existence of a positive feedback from PPARγ to C/EBPβ by a second independent method, we used the PPARγ activators rosiglitazone and pioglitazone to directly activate endogenous PPARγ in the absence of other stimuli (Willson et al., 2001) (Figure 2C). Addition of both activators induced a marked upregulation of C/EBPβ (Figure 2C, right panel). Finally, we also overexpressed PPARγ using retroviruses to confirm that increasing PPARγ expression resulted in increased C/EBPβ expression. The images of the single-cell analysis, as well as a quantitative scatterplot of the single-cell results, show significant correlation between PPARγ and C/EBPβ expression (Figure 2D). We also confirmed the existence of the PPARγ-C/EBPβ feedback loop in the 3T3-L1 cell model (Figure S2D), which argued that this second feedback loop is a general mechanism for driving adipogenesis. Thus, as depicted in the scheme in Figure 3A, two consecutive positive feedback loops generate a bimodal distribution of high or low PPARγ, C/EBPβ, and C/EBPα activity early in adipogenesis.
Consecutive and Cooperative Induction of the PPARγ-C/EBPα Followed by the PPARγ-C/EBPβ Positive Feedback Loops
The existence of feedback loops from PPARγ meant that we could trigger the expression of C/EBPβ, C/EBPα, and PPARγ just by adding a PPARγ activator, without needing glucocorticoids or increased cAMP. To determine whether the regulatory steps that initially induce the expression of PPARγ, C/EBPα, and C/EBPβ were cooperative, as would be predicted for a bistable system, we titrated the PPARγ activator rosiglitazone into the media of undifferentiated OP9 cells and monitored the resulting protein expression levels after 48 hr (Figure 3B), a time point at which all three transcription factors showed maximal expression (Figure 2C). Consistent with the existence of a cooperative step in the induction of C/EBPβ, C/EBPα, and PPARγ, the stimulus-response curves all had sigmoidal shapes that could be best fit with Hill coefficients of ∼2.5.
Interestingly, the half-maximum response (EC50) for C/EBPβ expression was 4-fold higher than that for C/EBPα expression, indicating that the PPARγ-C/EBPβ feedback loop has a higher threshold for activation than the PPARγ-C/EBPα feedback loop. Because it takes time to build up the level of PPARγ, the positive feedback loop between PPARγ and C/EBPβ would be predicted to then engage with a delay after the PPARγ to C/EBPα loop. We confirmed that there is indeed a marked delay between engagement of the PPARγ-C/EBPα and PPARγ-C/EBPβ feedback loops. Western blot analysis performed at different time points after treatment with rosiglitazone showed that C/EBPα expression reached a maximal level within 24 hr (Figure 3C). However, maximal C/EBPβ expression was reached only after 72 hr.
Our analysis introduces a regulatory motif whereby a first feedback loop has to be engaged for a prolonged time period in order for a second feedback to be triggered that then carries the differentiation commitment process forward. A plausible result of such a second amplification by the PPARγ-C/EBPβ feedback loop is to create a sharper transition to the differentiated state. Indeed, when we used siRNA against C/EBPβ to suppress the second PPARγ-C/EBPβ feedback loop, the transition from low to high PPARγ expression with increasing amounts of rosiglitazone was more gradual and less robust compared to the sharp transition in the cells transfected with control YFP siRNA (Figure 3D). With the PPARγ-C/EBPβ feedback loop suppressed, a large population of the cells was unable to transition to the high PPARγ state, even when maximal doses of rosiglitazone were applied (Figure S3). Similar to other systems with multiple positive feedback loops (Brandman et al., 2005), the higher cooperativity generated by two consecutive feedback loops gives cells a stable off, as well as on, state (as described in Figure 2A). The stable off-state allows cells to reject short- or low-amplitude stimuli, which provides one of the key mechanistic ingredients for a sharp, all-or-none transition to a committed on-state.
To understand the respective roles of the different feedback loop components, we performed detailed siRNA-mediated perturbation experiments. Knockdown of C/EBPα resulted in an almost-complete suppression of PPARγ expression (Figure 3D), confirming that the PPARγ-C/EBPα feedback loop was essential for PPARγ expression (Rosen et al., 2002). Knockdown of C/EBPα also resulted in almost-complete knockdown of C/EBPβ expression (Figure 3E), showing that the PPARγ-C/EBPβ feedback loop required the presence of a functioning PPARγ-C/EBPα feedback loop and arguing for the sequential induction model depicted in Figure 3F: (1) cAMP and glucocorticoid signals initially drive C/EBPβ expression; (2) increasing C/EBPβ above a critical threshold then triggers the start of a positive feedback between PPARγ and C/EBPα that, after a time delay; (3) induces the second PPARγ-C/EBPβ feedback loop so that most of the cells transition into a terminally differentiated state. However, there was always a fraction of cells falling back to the basal state (Figure 1E), raising the question how cells regulate which fraction becomes locked in the differentiated state.
Characterization of a Late-Acting, Third Positive Feedback Loop between PPARγ and the Insulin Pathway
We observed that upregulating PPARγ activity increased insulin receptor expression (Figure 4A), which was not surprising since an earlier study had shown that C/EBPα can regulate insulin receptor expression and PPARγ and C/EBPα are in a positive feedback loop (Wu et al., 1999). To test whether the converse were true—that the insulin pathway could regulate PPARγ expression, thus creating a third feedback loop from PPARγ via C/EBPα—we used siRNA to knock down insulin receptor expression and carried out the standard adipocyte differentiation protocol. Indeed, insulin signaling is required to increase PPARγ expression 2-fold between days 2 and 3 (Figure 4B). However, during the first 2 days of adipogenesis, insulin signaling has only a small effect on C/EBPβ and PPARγ expression (Figures 4B and S4A), most likely due to the fact that the insulin receptor is strongly expressed only after day 2 (Figure 4C). As a control in the siRNA experiments, knockdown of the GR, which is needed to start differentiation, suppressed C/EBPβ and PPARγ expression already at day 1 (Figure 4B). These results confirm the existence of a third feedback loop between PPARγ and the insulin receptor that only engages with a delay after the induction of the C/EBPβ-PPARγ-C/EBPα dual-positive feedback system (Figures 3C and 4A).
This third feedback loop between PPARγ and the insulin pathway is not needed to trigger the bistable switch as evidenced by the fact that the switch triggers regardless if insulin is added under normal differentiation induction with glucocorticoids and cAMP (Figure 4D) and that rosiglitazone stimulation induces the switch even though no insulin is added (Figure S3). Furthermore, pAKT levels that can be used to monitor insulin signaling are never bimodal during adipogenesis (Figure 4E), in contrast to the bimodal expression of the switch components C/EBPβ, C/EBPα, and PPARγ (Figure 1D). As shown in Figure 4D, the main function of this third feedback loop between PPARγ and the insulin receptor is to amplify and boost PPARγ expression after the switch is made. Thus, the consecutive action of the three positive feedback loops generates a subpopulation of differentiated cells with persistently high PPARγ levels.
Insulin Signaling Controls Fat Accumulation in Differentiated Cells in a Graded Fashion
To directly determine the relationship between PPARγ expression and lipid accumulation, we used immunohistochemistry to measure both parameters in the same individual cells. Because insulin signaling is a main regulator of adipocyte metabolism, we further monitored the insulin receptor pathway by measuring the cellular intensity of p-AKT. The inset plot in Figure 4F (left) shows that the differentiated population with high PPARγ had on average ∼3-fold more lipid incorporated than the low PPARγ population, consistent with the interpretation that a persistently high level of PPARγ defines the differentiated adipocyte state. However, at the single-cell level, fat accumulation correlated only weakly with relative PPARγ expression (Figure 4F, left). This can be seen by the wide spread of BODIPY intensities when focusing on cells with high PPARγ. Thus, whereas PPARγ drives the differentiation process and marks differentiated cells, fat accumulation itself must be under control of another signaling pathway. Strikingly, the degree of lipogenesis in these differentiated cells with high PPARγ could be more closely predicted by the relative activity of p-AKT (Figure 4F, right), arguing that the strength of insulin-Akt signaling is the main determinant of how much fat is accumulated in an individual differentiated cell. The same data further show that this relationship between p-AKT and fat accumulation is graded, not bistable, which means that once the switch is made into the differentiated, high PPARγ state and high levels of insulin receptors are present, there is no threshold that needs to be overcome to accumulate fat. Weak insulin stimuli will already cause some fat formation, and increasing the insulin stimulus will proportionally increase the amount of fat synthesis in existing adipocytes. Of note, there was a small fraction of the total cell population that had high BODIPY intensity (Figure 4F, left), and also high p-Akt, whereas having low PPARγ expression. These cells may have alternative regulatory mechanisms to increase insulin signaling not involving PPARγ expression.
Development of a Quantitative Molecular Model of Adipogenesis
We used the data from our single-cell analysis to generate a quantitative model of the C/EBPβ-PPARγ-C/EBPα-driven bistable switch, the insulin receptor-mediated PPARγ booster mechanism, and the subsequent insulin-regulated lipogenesis program. The diagram in Figure 5A illustrates the consecutive order of the three positive feedback loops that we identified for PPARγ activation (marked as steps 2, 3, and 4, respectively) and the subsequent insulin control of lipid accumulation (marked as step 5). Of note, there is likely no direct positive feedback between C/EBPα and C/EBPβ because chromatin immunoprecipitation data sets (Schmidt et al., 2011; Siersbæk et al., 2011) showed no evidence of C/EBPα or C/EBPβ binding to each other's promoters. In addition we also included in our model the previously demonstrated inhibition of the insulin signaling pathway by glucocorticoid and cAMP (Li et al., 2008), which we confirmed in experiments shown in Figures S4B–S4E. From a conceptual perspective the adipocyte differentiation system represents a novel design with consecutive positive feedbacks that engage at different times during the commitment process, allowing cells to ultimately reach a terminally differentiated state. The model recreates the initial increase in C/EBPβ expression, followed by the upregulation of C/EBPα and PPARγ, that is seen when thousands of single-cell measurements are averaged (Figure 5B, left; reproduced from Figure 1C).
To take into account that expression levels of regulatory proteins vary between individual mammalian cells (Niepel et al., 2009), we added stochastic variations to the relative amplitude of PPARγ, C/EBPβ, and C/EBPα synthesis, degradation, and basal expression parameters, respectively. As shown in Figure 5C when an average of 30% lognormal noise was added to the parameters, the model replicated the bimodality observed experimentally in Figure 1E, providing in silico evidence that the adipogenesis system is inherently bistable. No matter how we varied the initial parameters and pulled away from median values, the system always reverted back to one of the two stable points schematically shown in Figure 2A (right panel), which represent a stable differentiated and a stable nondifferentiated state.
This same analysis can also be used to estimate differences in the intrinsic noise among the PPARγ, C/EBPα, and C/EBPβ parameters. A best match was observed when PPARγ rates were varied less than the C/EBPβ and C/EBPα rates—by an average of 15% for PPARγ compared to 30% for C/EBPβ and C/EBPα, respectively (Figure S5A). The modeling further shows that if the protein variation would be much smaller, e.g., 3%, there would not be sufficient variation to create two populations of cells (Figure S5B). All cells would either remain undifferentiated, or all would switch to the differentiated state as the stimulus increases. On the other hand, if the variation were 100%, most cells would be in a state where the bistability of the system would break. The sweet spot in variation where bimodality is generated without breaking the system is approximately 15%–45%.
Thus, both experiments and modeling demonstrate that a uniform stimulus can create distinct differentiated and nondifferentiated subpopulations of cells with high versus low PPARγ/C/EBPβ concentrations, respectively. This induction of two clearly separate subpopulations can be explained by stochastic variation of the expression levels of the key regulatory proteins. Whether or not a particular "cell" will fall into the low or high PPARγ and C/EBPβ subpopulation depends on whether the relative expression levels of the regulatory proteins position the cell below or above a system's threshold where the bistable switch is triggered. Because they are connected by feedback, all three regulatory proteins contribute to setting the threshold of the system. However, consistent with a more central role of PPARγ in controlling the threshold, model calculations showed that expression of PPARγ immediately before the switch is triggered is more predictive of a cell's subsequent differentiation state compared to the levels of C/EBPβ or C/EBPα (Figure S5C).
Multiple Consecutive Positive Feedbacks Are Required to Create an Irreversible, Committed Differentiation State
We next tested whether the model reproduces our earlier observation in Figures 4B and 4D that the initial glucocorticoid and cAMP stimulation is sufficient to lock the system into a committed state even without the third positive feedback to the insulin receptor. The output of these simulations shows that PPARγ, C/EBPα, and C/EBPβ stayed high even after the glucocorticoid and cAMP stimuli were removed after 48 hr, demonstrating that the switch can be triggered even without the third positive feedback loop between PPARγ and the insulin receptor (Figure 6A). However, if the second feedback loop between PPARγ and C/EBPβ was removed from the model, PPARγ, C/EBPα, and C/EBPβ levels fell back down to their initial low values after glucocorticoid and cAMP stimuli were removed (Figure 6B). Plotting the steady-state curves (Figure 6B, right) showed that in a system with just one feedback loop, there is not much cooperativity, and thus, the steady-state curves do not have much sigmoidal bending. As a result, there are very few points in the parameter space where the steady-state curves can intersect and where the system can maintain two stable states.
To experimentally test whether or not a one-feedback loop model can trigger an irreversible transition, we used siRNA to knock down the expression of C/EBPβ versus control (YFP siRNA) and then added rosiglitazone to activate the feedback loops. As shown in Figure 6C, stimulating with rosiglitazone for 24 hr partially increased PPARγ. Then when the stimulus was removed for 24 hr, about half the cells locked into the differentiated, high PPARγ state, and the other cells fell back into the undifferentiated state. However, if the PPARγ-C/EBPβ is suppressed by siRNA knockdown, many cells could not sufficiently increase PPARγ, and even if they did, the majority fell back into the undifferentiated state when the stimulus was removed for 24 hr. As we show computationally and experimentally, the failure to maintain a committed state in a one-feedback loop system demonstrates why the increased cooperativity provided by the second positive feedback between PPARγ and C/EBPβ is of utmost importance in creating a robust bistable system.
History Dependence or Hysteresis of the Positive Feedback Loops
An important additional characteristic of a predicted bistable system is hysteresis. Hysteresis can be demonstrated by using an initial strong stimulus to lift cells into an on-state and then returning the cells to a low stimulus level that had previously kept the cells in the off-state. If a system has hysteresis, even though the stimulated cells have returned to a low stimulus level, they do not turn off. Rather, cells remain stuck in the on-state. A system with hysteresis thus has biochemical memory, and cells are capable of "remembering" that they have been stimulated even though the stimulus has been withdrawn. We observed this important hysteresis characteristic in our computational adipocyte differentiation model when we applied a transient pulse of glucocorticoid and cAMP (Figure 6A). Hysteresis was evident by the sustained elevation of PPARγ even after the stimulus was removed.
Figure 7A shows an experimental test for hysteresis in response to direct activation of PPARγ. A brief 3-hr-long pulse of PPARγ activity induced by adding rosiglitazone to the media, followed by a return to nonstimulated conditions, was sufficient to keep a subset of the cells in the high PPARγ state even after the stimulus was removed for almost 48 hr. The blue trace in Figure 7A is shown as a control where the cells were not subjected to the pulse of PPARγ activity, and no cell converted to the differentiated state even though the PPARγ activity was the same from 3 to 48 hr as for the rosiglitazone-pulsed cells. Figure 7B shows that the adipogenesis differentiation system is indeed capable of hysteresis, meaning that there is a discontinuous jump from the low-to-high PPARγ state. When all the cells in a well are averaged together, one observes a continuous sigmoidal curve (Figure 7B, left panel). However, as shown by the five inset plots in Figure 7B, each point in this curve is actually the average of a population of cells that is either in the low PPARγ state or high PPARγ state. If instead of averaging all the cells, one plots the percentage of cells in each population as a function of PPARγ activity, the discontinuity in the stimulus-response relationship becomes apparent with cells being in one of two possible PPARγ intensity states for a given intermediate stimulus (grey-shaded area in Figure 7B). However, when maximal rosiglitazone is applied for 48 hr, all cells switch into the high PPARγ state.
Consistent with the existence of hysteresis, increasing the stimulus duration locks more cells into the high PPARγ state (Figure 7C). When C/EBPα, C/EBPβ, and PPARγ levels were monitored in response to PPARγ activity pulses ranging from 3 to 48 hr (Figure S6), the fraction of cells that end up in the differentiated state gradually increases. Together with the amplitude dependence in Figure 7B, these results argue that both, the amplitude as well as the duration of the activation pulse, jointly control the probability of a cell transitioning to the high PPARγ differentiation state.
Finally, the observed hysteresis in Figure 7 demonstrates that the rosiglitazone-induced transition to the high PPARγ state transition does not bypass the bistable switch mechanism described in Figure 1E but rather induces the same circuit. Thus, the same bistable consecutive positive feedback circuit with hysteresis is induced by stimulation either with glucocorticoid and cAMP or by direct activation of PPARγ, further arguing that this triple feedback circuit is the core mechanism that converts preadipocytes to terminally differentiated adipocytes.
Three Consecutive Positive Feedbacks Drive Preadipocyte-to-Adipocyte Differentiation
Our results demonstrate that a commitment decision is made by preadipocytes early in adipogenesis before the appearance of lipid droplets, which has been a previous criterion for defining a terminally differentiated adipocyte state. This commitment process is bistable rather than graded. We demonstrated that the commitment decision relies on three consecutive positive feedback loops: a first loop between C/EBPα and PPARγ, followed by a second loop between PPARγ and C/EBPβ, and a third positive feedback between PPARγ and the insulin receptor. Importantly, we found that the second feedback loop back to C/EBPβ only engages at a higher PPARγ level. The requirement for higher PPARγ caused a marked delay in the activation of the PPARγ-C/EBPβ loop compared to the PPARγ-C/EBPα loop. A third positive feedback loop between PPARγ and the insulin receptor then further boosts PPARγ expression and helps to maintain and consolidate the terminally differentiated state. This third positive feedback only engages after an additional delay forced by the need for insulin receptors to be expressed at a higher level and by cAMP and glucocorticoid suppression of the insulin signaling pathway, which is only removed late in the differentiation process (Figure S4). Together, the successive triggering of three positive feedbacks forces a sequence of predefined events onto the adipocyte differentiation process.
We demonstrated that the same differentiation switch can be induced by either the glucocorticoid and cAMP-mediated induction of C/EBPβ or, more directly, by the rosiglitazone-mediated activation of endogenous PPARγ. Both stimuli show hysteresis, have the same bimodality in the induction of the high C/EBPβ and PPARγ state, and have the same consecutive order of activation (Figure 7). The identical consecutive activation by the two different stimuli argues that the same feedback circuit design is responsible for the endogenous, as well as drug-induced, differentiation of adipocytes. This has mechanistic implications, suggesting that the same consecutive positive feedback loop circuit design can be triggered by different physiological or drug-induced stimuli, arguing that the circuit we identified is the core module responsible for fat cell differentiation.
The rosiglitazone experiments in Figure 7 demonstrated that the PPARγ-C/EBPα-C/EBPβ bistable switch can sense and transduce both the duration, as well as the amplitude, of the activating pulse into differentiating an increasing fraction of the cells while rejecting weak stimuli. This bistable switch provides a stable off-state that allows preadipocytes to exist for long periods of time in an undifferentiated state as long as the stimuli that activate PPARγ stay below a critical threshold and helps to explain how only a small fraction of adipocytes are renewed in an adult human every year (Spalding et al., 2008). In contrast, when the inducing stimuli are above the threshold, the fraction of cells converted to adipocytes can be controlled in a graded fashion (over about a factor of 16 in rosiglitazone in Figure 7B), allowing for better control of the number of adipocytes than would be obtained in a system in which all preadipocytes convert to adipocytes in an all-or-none fashion once a single critical threshold is crossed.
How can one explain why only a part of the cell population converts to the differentiated state when stimuli have submaximal amplitude and/or duration? If one assumes that the cells are identical and that differentiation is an all-or-none process at the level of single cells, all cells should differentiate for stimuli above a particular threshold value or all cells should remain undifferentiated for stimuli below that threshold value. As shown in Figure 7B, there is a range of low-amplitude stimuli where no differentiation is observed, followed by a range of intermediate-amplitude stimuli (shaded in grey) where the fraction of cells that differentiates increases in a graded fashion, followed by a range of high-amplitude stimuli where all cells are converted to the high PPARγ differentiated state. How can we reconcile an all-or-none differentiation switch with the observed graded response in the grey box? As demonstrated by model calculations for differentiation induced by glucocorticoid and cAMP stimuli (Figure 5C), the partial conversion of a cell population can be explained by cell-to-cell variability in expression levels of regulatory components, which results in variable sensitivity to PPARγ within an otherwise homogenous population of cells. Thus, for a given submaximal stimulus concentration, some cells will convert to the high PPARγ state sooner than others. The same fractional conversion also applies to increases in the duration of maximal stimuli (Figure S6).
Together, these results argue that organisms employ a system that combines consecutive positive feedback and stochastic variation and then use both the amplitude and the duration of the activating stimulus to control the number of differentiated adipocytes. The demonstrated requirement for persistent and strong inductive signals confers robustness to the system by preventing short- and low-amplitude stimuli from accidentally triggering differentiation.
Graded Control of Fat Accumulation in Individual Differentiated Adipocytes by Insulin
A consequence of this irreversible commitment step early in differentiation is that cells must exist that are already committed to becoming fat cells but do not yet have a visible increase in fat storage. Once they switch into the persistently high PPARγ state, these differentiated adipocytes control the degree of lipogenesis in a graded fashion with fat accumulation closely correlating with the strength of insulin pathway activity in each cell (p-AKT; Figure 4). In contrast the relative level of PPARγ, which can vary in the differentiated cells, only weakly correlates with fat accumulation. Thus, two important regulatory programs need to be distinguished in the management of the weight of mammals: the regulation of the degree of fat storage in individual adipocytes, and the total number of adipocytes per organism. This total number of adipocytes changes only slowly with about 10% turnover per year in humans (Spalding et al., 2008). Our study then argues that a primary role for the insulin pathway is to directly control how much fat is stored in individual cells rather than regulating the differentiation decision. In contrast, the differentiation decision, which controls the number of fat cells, is under a mixed control of glucocorticoid and cAMP stimuli with only a minor contribution from insulin signaling.
A Computational Molecular Model for Adipocyte Differentiation
In order to mechanistically understand the differentiation regulatory circuit, we developed a computational model for the conversion of preadipocytes to adipocytes. Specifically, our goals in modeling were to (1) learn more about why cells use a consecutive multipositive feedback circuit design to control differentiation, and (2) understand our experimental observation of bimodality and how submaximal stimuli could convert only a fraction of stimulated preadipocytes to adipocytes. As a third, more long-term goal, we were interested to use differentiation models to predict which regulatory inputs are best suited as drug targets to regulate the total number of human adipocytes.
We showed that a single positive feedback loop with low cooperativity can amplify signals but cannot generate the observed robust bistability. The experimentally identified consecutive feedbacks make the differentiation process more nonlinear (ultrasensitive) and the switch more robust. Our model explains and quantitatively recapitulates how these sequential feedback loops are engaged to drive the preadipocytes into a persistent state characterized by elevated PPARγ, C/EBPα, C/EBPβ, and insulin receptor expression. This example of a differentiation process provides a conceptual framework that a sequential positive feedback circuit design is well suited to induce a robust transition to a differentiated state. It is suggestive to propose that similar consecutive positive feedback circuit designs drive many, if not most, other differentiation processes.
Our model further demonstrated how stochastic variations in the expression levels of C/EBPβ, C/EBPα, and PPARγ cause a differentiating and nondifferentiating population to coexist even though all cells are subjected to the same stimuli. This finding from the model can explain in molecular terms why intermediate stimuli only convert a fraction of preadipocytes into differentiated cells rather than generating potentially undesirable and misfunctioning partially differentiated cells. This single-cell variation concept also provides a molecular explanation of how organisms can have robust, all-or-none conversion of individual preadipocyte cells into adipocytes, whereas at the same time ensuring that the total population of preadipocytes does not convert in an all-or-none fashion for intermediate stimuli. Rather, single-cell variation ensures that only a small fraction of the total preadipocyte population converts everyday to an irreversible, differentiated adipocyte state (Spalding et al., 2008).
Finally, understanding if, when, and where a clear commitment decision is made by preadipocytes is critical for knowing how to target therapeutics to manipulate the differentiation process as a possible means to treat obesity, diabetes, and other adipocyte-associated diseases. Because the signaling and transcriptional network controlling adipogenesis involves multiple feedbacks with different time constants, it cannot be readily understood by a graphical diagram alone. We argue that our quantitative molecular working model of adipogenesis can be used to guide experiments, to conceptually understand the induction process, and also to have a new way to test the effect of different inputs and perturbations to components in the network. As an example of such a use of the model to predict outcomes, our experiments in Figure 7 confirmed the prediction from the model that direct activation of PPARγ by rosiglitazone should induce the bistable switch with coinduced C/EBPα, PPARγ, and C/EBPβ expression rather than bypassing the switch and directly regulating the adipogenesis-relevant genes downstream of PPARγ. The latter could have been predicted with equal plausibility. Instead, our study demonstrated that direct PPARγ activation by rosiglitazone triggers the same consecutive positive feedback circuit that is triggered by glucocorticoid and cAMP signaling. The ability of the model to predict drug action exemplifies a future use of such differentiation models to predict optimal combinations of potential therapeutic interventions to control the total number of adipocytes in a patient by increasing or decreasing the rate of differentiation from preadipocytes to adipocytes.
Because of low cooperativity in typical single transcriptional feedback loops, multiple feedback loops are required in adipogenesis to generate sufficient cooperativity to reliably convert to the differentiated state. Importantly, the circuit design identified here with consecutive positive feedback loops, including one that reaches back to C/EBPβ, ensures that the differentiation decision is not triggered accidentally by uncoordinated or brief and low-amplitude hormonal stimuli. Our model and experimental analysis further show how stochastic variation in the expression of regulatory proteins is sufficient to explain how a submaximal stimulus triggers an all-or-none terminal differentiation of only a fraction of proliferating precursor cells. Together, our study provides conceptual insights into the adipogenesis process that likely applies to many, if not most, cell fate decisions.
Experimental Procedures
Cell Culture, Differentiation, Transfection, Antibodies, and Plasmids
OP9 and 3T3-L1 cells were cultured according to the protocols in Wolins et al., 2006. OP9 cells were grown in growth medium consisting of MEM-α, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin, plus 20% FBS. 3T3-L1 cells were grown in 3T3-L1 propagation medium: DMEM with 10% bovine calf serum, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. To induce differentiation, confluent cells were treated with a differentiation medium containing growth medium plus the standard adipogenic cocktail (DIM): 1 μM dex, 175 nM insulin, 0.5 mM IBMX, and 10% FBS. After 48 hr, the differentiation medium was replaced with growth medium, plus 175 nM insulin and 10% FBS. Diced pool siRNA was generated as previously described by Galvez et al., 2007 and transfected into OP9 cells using RNAiMax (Invitrogen) and a reverse-transfection protocol. DNA transfection was carried out by retroviral infection. siRNA specificity was verified using a second diced pool of siRNA, as well as with synthetic siRNA (see Figures S2A–S2C). Sources for antibodies, reagents, constructs, and primers are provided in the Extended Experimental Procedures.
Automated Image Acquisition and Processing
Images were acquired on an ImageXpress 5000A automated epifluorescence microscope (Molecular Devices, Sunnyvale, CA, USA) using a 4X Plan Fluor objective and a 1,280 × 1,024 pixel, cooled CCD camera with a 12-bit readout. Image analysis was performed using custom software written in MATLAB. In brief, nuclear centroids were identified in images of Hoechst stain. A nucleus mask was generated for each cell by expansion from the centroid to reach 30% of maximum intensity. A cell mask was then generated by expansion of the nucleus mask 7 μm to include both the nucleus and the perinuclear region. After local background subtraction, the nucleus mask was used to measure PPARγ, C/EBPβ, and C/EBPα mean intensities, and the cell mask was used to measure BODIPY (lipid droplet content) and p-AKT mean intensities.
Computational Model
MATLAB SimBiology was used to program and run the model simulations. The model equations used to generate Figures 5B and 5C are presented below. Additional details can be found in the Extended Experimental Procedures.
The model has three inputs: [IR], [GR], and [cAMP].
siRNA Transfection
Synthetic SMARTPOOL siRNA for PPARγ, C/EBPβ, and C/EBPα were purchased from Dharmacon. To generate diced siRNA, gene-specific primers were designed with an in-house primer program and were used to generate ∼600 bp cDNA fragments immediately upstream of the stop codon of each mRNA by PCR. An additional set of nested primers was designed to add T7 promoters at both ends of the final cDNA fragment. Nested PCR products were subjected to in vitro transcription, in vitro dicing, and purification to produce siRNA as described previously (Galvez et al., 2007). The OP9 cells were transfected with siRNA by a reverse transfection protocol. For each 96-well well, 2 pmol of diced-pool siRNA was diluted in 10ul of Opti-Mem I Medium. 0.2ul of RNAiMax (Invitrogen) diluted in 10ul of Opti-Mem I was then added, mixed well, and then incubated for 20 min at room temperature. This mixture was then placed into a 96-well, and OP9 cells were added (15,000 cells suspended in 80 μl of growth medium without antibiotics). After 24 hr, the media was replaced with differentiation media to induce differentiation following differentiation method. Specificity for all siRNA used in the paper - PPARγ, C/EBPβ, C/EBPα, glucocorticoid receptor, and insulin receptor – was verified by demonstrating that the same results were obtained using two different diced pools of siRNA each targeting a different coding region of the respective gene (Figures S2B and S2C). Specificity of PPARγ, C/EBPβ, C/EBPα siRNA was also confirmed using commercially-obtained synthetic siRNA.
Retroviral Infection of Cells
HEK293T were transfected in 6-well wells at 30% confluence using Fugene6 and 1 μg of PCL-ECO packaging construct together with either 1 μg of pBMNi-PPARγ-IRES-hcRed, pBMNi-C/EBPα-IRES-GFP, or pBMNi-IRES-GFP expression plasmids. 4 ml of viral supernatant was collected both at 48 and 72 hr after transfection. The 8 ml total of viral supernatant was combined, concentrated using PEG-it virus precipitation solution (System Biosciences), and resuspended in 100ul of PBS to make concentrated retrovirus. 5000 OP9 cells in 100ul of OP9 growth media were plated into each 96-well well 12 hr before infection. 5 ul of concentrated retrovirus plus polybrene (8mg/ml final concentration) was added per 96-well well. After 12 hr, the media in each well was changed to fresh OP9 growth media. Cells were fixed 10 days after infection.
Immunofluorescence Staining
OP9 cells were fixed with 3% paraformaldehyde in PBS for 30 min. Then the cells were gently washed 3X with PBS and permeabilized with 0.05% saponin (Sigma #47036), blocked with 3% bovine serum albumin (Sigma #7906) and stained with anti-PPARγ (1:500 Santa Cruz Biotech #sc-7273), anti-C/EBPα (1:500, Santa Cruz Biotech #sc-61), anti-C/EBPβ (1:500, Santa Cruz Biotech #sc-150), anti-C/EBPβ (1:500, Santa Cruz Biotech #sc-7962), anti-pAKT(S473)(1:1000, Cell Signaling #4060) and BODIPY 493/503 (1ug/ml, Molecular Probes #D-3922). Alexa Fluor-514 (#A31558), 555 (#A21429), 594 (#A11032) and 647 (#A31571) (1:1000, Invitrogen) were used as secondary antibodies.
Western Blot Analysis
Cultured cells were lysed using the NE-PER nuclear and Cytoplasmic extraction kit (Pierce; Rockford, IL) for nuclear and cytoplasmic lysate. Protein concentrations were determined by BCA protein assay kit (Pierce) with BSA as standard. Gel electrophoresis was performed using precast Bis-Tris 4%–12% gradient polyacrylamide gels with NuPAGE buffer system (Invitrogen). Proteins were transferred to PVDF membranes (Immobilon-FL,Millipore) using the Xcell II Blot Module (Invitrogen). The membranes were blocked for 1 hr in Odyssey blocking buffer (Li-Cor biosciences, Cambridge, UK) and were probed with the following primary antibodies: PPARγ, C/EBPα, C/EBPβ and Insulin receptorβ (1:500, Santa Cruz Biotechnology, Inc.), aP2/FAPB4 (1:3000, Cell Signaling Technology), adiponectin and perilipin (1:1000, Abcam). Detection was performed using a secondary antibody (1:3000) coupled to an IR 680 or IR 800 dye, and the membranes scanned using the Li-Cor Odyssey IR imager.
Model Description
The fixed parameters used were: degCEBPB = 0.1 (rel unit), degPPARG = 0.15, degCEBPA = 0.2, degFAT = 0.06, degpAKT = 0.5, synCEBP = 0.075, synPPARG = 0.7, synCEBPA = 0.7, synFAT = 0.25, synpAKT = 0.08, alpha1 = 1.0, alpha2 = 1.0, alpha3 = 1.0, alpha4 = 1.5, alpha5 = 0.5, BasePPARG = 0.06, BaseCEBPA = 0.06, BaseCEBPB = 0.05, alpha6 = 0.6, alpha7 = 0.1, BaseIR = 0.005, alpha8 = 1.0, synIR = 0.6, degIR = 0.05.
Initial values for the three inputs: insulin, [IR] = 0.25 (rel. unit); [cAMP] = 2.0; glucocorticoids. [GR] = 2.0.
Specific details of each equation:
(2) d[PPARγ]/dt: Because they can bind to the same DNA sequences and can replace each other at binding sites, C/EBPα and C/EBPβ are added together in the term reflecting the positive feedback between these transcription factors (TFs) and PPARγ. The pAKT term reflects the positive feedback between the insulin signaling pathway and PPARγ. There is no Hill coefficient in the pAKT feedback because our experiments show that pAKT signaling is always graded and never bimodal.
(3) There are two main mechanisms of cooperativity in this system: 1) C/EBPβ and C/EBPα need to dimerize to activate transcription and 2) there are multiple binding sites for C/EBPα and C/EBPβ on the PPARγ promoter and multiple binding sites for PPARγ on the C/EBPβ and C/EBPα promoters (Lefterova et al., 2008; Mikkelsen et al., 2010; Steger and Lazar, 2011). Thus Hill coefficients of at least 2 were chosen for the feedback loops in the first 3 equations. Given that the precise cooperativity is not known and is difficult to measure experimentally, we chose the respective Hill coefficients because lower cooperativity did not give sufficient bistability to match the experimental data. In the model, we used the minimal cooperativity needed to recapitulate the experimental data.
(4) d[pAKT]/dt: pAKT is a readout of the insulin pathway activity. pAKT is inhibited by [cAMP] and [GR] stimulation.
(5) d[IR]/dt: C/EBPα upregulates IR expression.
(6) d[Fat]/dt: The amount of fat is dependent on both AKT activity (pAKT) and PPARγ expression. However, the constants alpha7 and alpha8 were shown to reflect that the amount of fat is much more strongly dependent on pAKT than on PPARγ expression.
(7) Both PPARγ and C/EBPα bind to their own promoters respectively (Lefterova et al., 2008; Steger et al., 2010) which suggests that these transcription factors could be regulated by autofeedback loops. We did not include autofeedback for PPARγ or C/EBPα because there is no experimental evidence yet in the literature that they are regulated by autofeedback. Even though binding sites are present, one cannot assume actual transcriptional regulation without testing for this. An additional reason why we did not include autofeedback for PPARγ is that our experimental results (Figure 3E) showed that knockdown of C/EBPα and subsequent activation of PPARγ resulted in an almost complete reduction of PPARγ expression, indicating at best only a minor role of an autofeedback PPARγ loop if it does prove to exist. We did not include any autofeedback term in the C/EBPβ equation in the model since no evidence has been shown yet even in large-scale CHIP studies that C/EBPβ binds to its own promoter (Siersbæk et al., 2011; Steger et al., 2010).
(8) Small random variations were introduced to model parameters assuming log normal distribution of the parameters.
(9) All models presented in this manuscript will be uploaded to the EMBL-EBI BioModels repository: (http://www.ebi.ac.uk/biomodels-main/).
We would like to acknowledge Tobias Meyer, Sabrina Spencer, Graham Anderson, Brian Feldman, Fredric Kraemer (Stanford), Jake Lusis (UCLA), as well as the members of the M.N.T. lab for helpful discussions and careful reading of the manuscript. We gratefully acknowledge funding from Stanford University and the Stanford Cardiovascular Institute (to M.N.T.) and DFG AH 220/1-1 (to R.A.).
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Supplemental References
Galvez et al., 2007 Galvez, T., Teruel, M.N., Heo, W.D., Jones, J.T., Kim, M.L., Liou, J., Myers, J.W., and Meyer, T. (2007). siRNA screen of the human signaling proteome identifies the PtdIns(3,4,5)P3-mTOR signaling pathway as a primary regulator of transferrin uptake. Genome Biol. 8, R142..
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Publication Information
Received: May 14, 2012
Revised: August 10, 2012
Accepted: August 31, 2012
Published online: October 11, 2012


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[JURIST] The US Court of Appeals for the Eighth Circuit on Tuesday unanimously upheld [ruling, PDF] a 2006 federal court ruling [PDF text; JURIST report] that the state of Missouri must provide inmates with transportation to a medical facility for a non-therapeutic abortion. The Eighth Circuit rejected the trial court's conclusion that a non-therapeutic abortion constitutes a "serious medical need," and therefore may not be intentionally denied by the state under the Eighth Amendment. The Eighth Circuit instead affirmed the judgment under an analysis of the Fourteenth Amendment established by the US Supreme Court's 1987 case Turner v. Safley [text], which states that "prison regulations restricting constitutional guarantees are valid only if the regulations are 'reasonably related to legitimate penological interests.'" The Missouri Department of Corrections [official website] will comport with the decision as it considers an appeal.The American Civil Liberties Union applauded the decision [press release] as "consistent with rulings from across the country that women prisoners do not lose their reproductive rights once they are incarcerated." The Kansas City Star has more.
[JURIST] An Afghan journalism student charged with blasphemy was sentenced to death Tuesday, following a recommendation from the Afghan Council of Mullahs. A three-judge panel found that papers printed from the Internet by 23-year-old Sayad Parwez Kambaksh, allegedly including discussion of women's role in society, insulted Islam and invoked Article 130 of the Afghanistan Constitution [text], allowing for execution consistent with Hanafi [GlobalSecurity backgrounder] law. Kambaksh showed the papers to a university teacher and classmates, some of whom said Kambaksh wrote the document himself. It was not clear if Kambaksh had legal representation at the trial, and his trial date was not made public. He will remain in prison as the sentence is appealed.Kambaksh has received public support [RWB report] from Reporters Without Borders [advocacy website], as well as European Parliament President Hans-Gert Pöttering [official website]. On Monday, Afghan prosecutor Hafizullah Khaliqyar said Kambaksh supporters would be imprisoned. AP has more.
[JURIST] The Council of Europe's Parliamentary Assembly (PACE) [official website] Tuesday released a report [PDF text; CoE press release] criticizing the United Kingdom's voting system for its vulnerability to fraud. A February 2007 fact-finding trip conducted by a PACE committee found that fraud risks are "mainly the result of the, rather arcane, system of voter registration without personal identifiers." UK voting registers currently list only the voters' names and addresses and do not include such "personal identifiers" as date of birth or signatures. The report said that UK elections are "conducted democratically" and, while not recommending a monitoring procedure, the report said:
The Monitoring Committee should, in its periodic reports on the honouring of commitments by member states, pay special attention to electoral issues in the United Kingdom and, if the vulnerabilities noted are found to undermine the overall democratic nature of future elections in Great Britain, apply to initiate a monitoring procedure with respect to the United Kingdom.
[JURIST] Jury selection began Tuesday in the retrial of six of the seven men charged with allegedly conspiring to bomb [DOJ press release] the FBI headquarters in Miami and the Sears Tower in Chicago. The seven men first went to trial last year, but in December one man was acquitted and a mistrial was declared [JURIST report] for the other six after the case ended in a hung jury. If convicted, each defendant faces up to 70 years in prison. The seven were indicted [JURIST report] in 2006 on charges [indictment, PDF] of conspiring to provide material support to al Qaeda; conspiring to provide material support, training, and resources to terrorists; conspiring to maliciously damage and destroy by means of an explosive; and conspiring to levy war against the government of the United States. The indictment alleges that ringleader Narseal Batiste recruited the six other defendants to "organize and train for a mission to wage war against the United States government," and that they pledged an oath to al Qaeda in an attempt to secure financial and logistical backing. Lawyers for some of the men said that their clients were entrapped [JURIST report] by an FBI informant posing as an al Qaeda operative. AP has more.
[JURIST] Jose Padilla [JURIST news archive] was sentenced to 17 years and four months in prison Tuesday in connection with his conviction [JURIST report] on terrorism-related charges. US District Judge Marcia Cooke [official profile] handed down the sentence following two weeks of sentencing hearings, during which the government sought life sentences for Padilla and his two co-defendants, Adham Amin Hassoun and Kifadh Wael Jayyousi [GlobalSecurity profiles]. The defense team raised over 90 objections to a prosecutorial report supporting the request for life sentences, arguing it misrepresented the evidence presented at trial; Padilla's lawyers argued for a 10-year sentence, while lawyers for Hassoun and Jayyousi asked for penalties of four to six years and probation, respectively. Hassoun was sentenced to 15 years and eight months while Jayyousi received a 12-year, eight-month sentence. Cooke ruled last week that she could apply enhanced federal terrorism penalties [JURIST report] in the case, which in effect permitted her to consider the death penalty. Padilla, Hassoun and Jayyousi were convicted in August 2007 of conspiracy to commit illegal violent acts outside the US, conspiracy to provide material support to terrorists, and providing material support to terrorists. Padilla, a US citizen, was arrested in 2002 at Chicago's O'Hare International Airport and subsequently detained as an "enemy combatant" [JURIST news archive] at a Navy military brig in Charleston, South Carolina. Initially alleged to have planned the explosion of a "dirty bomb" in the United States, Padilla went from enemy combatant to criminal defendant when he was finally charged in November 2005 and transferred to civilian custody [JURIST report] in January 2006. AP has more.
[JURIST] Kenya's main opposition party, the Orange Democratic Movement (ODM) [party website], has filed a formal complaint at the International Criminal Court (ICC) [official website], alleging that the Kenyan government has committed crimes against humanity in using force against demonstrators protesting the recent disputed re-election [JURIST report], an ODM spokesperson said Tuesday. The violence began after the re-election of Kenyan President Mwai Kibaki [official profile], who has has long been accused of using his position to favor members of the Kikuyu tribe. Over 700 people have died since protests began last month, prompting Human Rights Watch to urge the government to ban police from using excessive and lethal force against protesters [press release]. Thirteen nations, including several European Union members and the United States, have threatened to cut off aid to Kenya's government until the crisis is resolved.The controversial presidential vote has sparked simmering ethnic tensions in Kenya [JURIST news archive]. Kibaki won the election despite early opinion polls that placed rival ODM candidate Raila Odinga in the lead. Thousands of opposition supporters have continued calls for protests against the elections [JURIST report], despite the government's temporary ban on public rallies. The ODM has also threatened to use economic boycotts and strikes [JURIST report] to continue their protests. AFP has more.
[JURIST] The US Supreme Court [official website; JURIST news archive] ruled Tuesday that a prison inmate cannot bring a lawsuit against federal prison guards over the handling of the inmate's possessions under the Federal Tort Claims Act [text]. In Ali v. Federal Bureau of Prisons [Duke Law case backgrounder; JURIST report], Abdus-Shahid Ali sued prison officials working for both the District of Columbia Department of Corrections and the US Bureau of Prisons, arguing that they illegally confiscated his religious property, including copies of the Koran and his prayer rug, and harassed him for expressing his Muslim faith while in custody. The Federal Tort Claims Act includes an exception [28 USC 2680(c)] barring claims for detained property against "law enforcement officers," but Ali argued that the exception applies to officials working in an excise or customs capacity. The Supreme Court rejected that argument and affirmed the Eleventh Circuit's opinion [PDF text] in the case, concluding "that the broad phrase 'any other law enforcement officer' covers all law enforcement officers." Read the Court's opinion [text] from Justice Thomas, along with a dissent [text] from Justice Kennedy and a second dissent [text] from Justice Breyer. AP has more.
[JURIST] The European Court of Human Rights (ECHR) [official website] Tuesday ruled [DOC text; press release] that France violated the rights of a lesbian woman by refusing her application to adopt a child because of her sexual preference. The French officials rejected the woman's application on the grounds that she and her partner were not able to present a male "referent," even though French law permits single adults over the age of 28 to adopt children. The ECHR ruled that France violated the petitioner's rights under Article 8 of the European Convention on Human Rights [PDF text], which guarantees respect for private and family life, and under Article 14, which prohibits discrimination based on sexual orientation. The ECHR ordered the French government to pay 24,528 in non-pecuniary damages, costs and expenses. AP has more.
[JURIST] The British Columbia Civil Liberties Association (BCCLA) [advocacy website] on Monday released internal Canadian government documents [PDF text] detailing evidence of continued mistreatment and abuse of detainees transferred by Canadian forces to Afghan authorities. The documents, originally distributed to senior officials of the Canadian government and officers of the Canadian military, detail an investigation conducted by Canadian officials last November which found circumstantial evidence that detainees were abused at a facility belonging to the Afghan National Directorate of Security in Kandahar. Last November, the Federal Court of Canada ruled [JURIST report] that Amnesty International [advocacy website] and the BCCLA should be granted public interest standing to seek judicial review of actions or potential actions of Canadian military personnel deployed in Afghanistan, rejecting the Canadian government's motion to strike the groups' application on the grounds that they lacked standing and the issue was political in nature. The rights groups allege that Canadian forces deployed in Afghanistan are bound by the Canadian Charter of Rights and Freedoms [text] and that Canadian personnel transferring Afghan prisoners at risk of torture by Afghan authorities have violated the Charter's prohibition against the deprivation of life, liberty, and security. Also in November, Canadian opposition parliamentarians called for the government to stop detainee transfers [JURIST report], after documents [text] released by the Canadian government appeared to confirm allegations that transferred suspects had been subjected to abuse. Last May, Canada and Afghanistan entered into an agreement allowing for the monitoring of prisoners [JURIST report] transferred from Canadian to Afghanistan custody. The Globe and Mail has more.
[JURIST] Zimbabwean police banned an opposition party political march on Tuesday, setting the stage for a legal battle involving recent amendments [JURIST report] to Zimbabwe's restrictive Public Order and Security Act [PDF text], which allows police to deny any party's application for a public gathering. Under the old version of the law, the Minister of Home Affairs, which is controlled by the ruling party, had exclusive jurisdiction over appeals of such denials, but under amendments signed by President Mugabe [JURIST report] on Sunday, political parties may appeal to a neutral magistrate and police may justify permit denials only on security grounds. The opposition is expected to appeal the denial Tuesday. The march had been scheduled for Wednesday. AP has more.Mugabe is seeking a sixth term as president of Zimbabwe, and he is expected to prevail over splintered opposition. In the past, Zimbabwe has used the Public Order and Security Act to crack down on anti-government protests. In May, police in Harare arrested [JURIST report] members of the National Constitutional Assembly [official website] who protested against proposed amendments to the Zimbabwean Constitution [PDF text], later passed and signed into law [JURIST report], which essentially allow Mugabe to pick his successor. That same month, Zimbabwean police broke up a rally of about 50 lawyers who had gathered to protest the the arrest and imprisonment of two human rights advocates; the lawyers argued that the Public Order and Security Act exempted them from a general ban against demonstrations [JURIST reports].
[JURIST] The Russian Prosecutor General's office [official website, in Russian] said Tuesday it has launched a forgery investigation targeting former Russian prime minister and now opposition leader Mikhail Kasyanov [BBC profile]. According to Russian news agencies, the investigation relates to allegations that signatures were forged on petitions nominating Kasyanov for the upcoming March 2 Russian presidential elections. A spokesperson for Kasyanov's campaign immediately characterized the investigation as politically motivated.Kasyanov and fellow opposition leader Garry Kasparov [personal website, in Russian] have been strong critics of current Russian President Vladimir Putin [official website] and his allies in the run-up to the March vote. Putin dismissed Kasyanov, a close associate of former president Boris Yeltsin, as head of government in February 2004 after a four-year tenure. Last November, Kasparov was detained for five days for organizing an unsanctioned protest [JURIST report] against Putin's policies and resisting arrest. AP has more.
[JURIST] The government of Pakistan, which has kept Pakistani Supreme Court Chief Justice Iftikhar Mohammad Chaudhry [JURIST news archive] and several other judges and lawyers under preventative detention since President Musharraf declared emergency rule [text; JURIST report] on November 3, may be attempting to avoid constitutional restraints on the duration of those detentions, according to a Tuesday report [text] by Pakistan's The News daily. According to The News, the constitution requires that in order for preventative detention to be extended beyond 90 days, a review board must hear the cases and decide whether to extend the detention. The 90-day period for Chaudhry and other detainees expires January 31. Thus far, the government has not referred the cases to the review board. Rather, the government has explained that Chaudhry and the other deposed judges are not under detention at all, as they are not being held pursuant to a court order, and thus do not qualify for review. As for the lawyers, it is the government's position that they are in the custody of provincial governments, not the federal government, and thus do not qualify for review. Clause 4 of Article 10 of the Constitution of Pakistan [text] says:
No law providing for preventive detention shall be made except to deal with persons acting in a manner prejudicial to the integrity, security or defence of Pakistan or any part thereof, or external affairs of Pakistan, or public order, or the maintenance of supplies or services, and no such law shall authorise the detention of a person for a period exceeding [three months] unless the appropriate Review Board has, after affording him an opportunity of being heard in person, reviewed his case and reported, before the expiration of the said period, that there is, in its opinion, sufficient cause for such detention, and, if the detention is continued after the said period of [three months], unless the appropriate Review Board has reviewed his case and reported, before the expiration of each period of three months, that there is, in its opinion, sufficient cause for such detention.
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text:
This is a Step-by-Step Instructable on how to grow bell or chili peppers from your favorite store bought varieties! And regardless of colour, flavor, or size, all peppers are grown pretty much the same way making this is a very fun, cheap, and easy to do project that I'm sure anyone of any age would enjoy doing!
So read on and learn how to grow some of these for yourself!
PS, i know the intro image is awful, no need to remind me xD
Step 1: Materials
(I used for this instructable but they're not necessary)
+Peppers or Pepper Seeds
- Any variaty of pepper will work
- If getting seeds, i wouldn't recommend getting hybrids, they are very simple to grow and are very resilient and strong, but seeds harvested from these peppers will likely be inferior to the origional plant. They will lack the "vigor" they had before.
All of these are optional.
+ A seed germination set-up
+ Soil (Sphagnum Moss, Cheap Soil, Quality Soil --mixed)
+ Plastic Baggies
+ Brown Paper Bag
+ and some Pots!
The round planter has a "Trinidad Scorpion Butch T" and in the long one I just threw in some packets of crushed chili from a fast food vendor as an experiment. I understand these packets are usually from Cayenne. Has anyone tried this before with any luck? If they're radiated when processed, I suspect they're all dead and will not sprout.
I have several more 1 inch tall "Trinidad Scorpion Butch T" plants growing inside that I plan on growing in a "Topsy Turvy® Hot Pepper Planter" when they get a little bigger.
happy gardening and post again
angel x
You might have more luck with this technique with fruits from a farmer's market. It would also be much easier to get in contact with the farmer to find out about varietal information.
Have you gotten any fruit from your plants yet? How did they turn out? Did you get lucky?
Source: http://groups.yahoo.com/group/cnyplantcycle
I love peppers but they never do well in my garden, unless I feed them so much stuff that I may as well get store bought. Then they are never as big. I am in zone 6.
I just bought Habanero seeds that were freakin' expensive... I hope that I can save the money next year by propagating them!
Yes, green pepper will turn red, but a red pepper has a very different taste.
I usually buy organic veggies when I try to extract seeds for drying.
Not a guarantee but I usually get the good ones.
Nice peppers,
Mine are not always as nice, peppers are a small challenge. 8)


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text:
Release Notes: Support for importing servers from iTunes PCAST files was added. A cleanup_days bug was fixed (if a command-line option was not set, it overrode the default with a null value). BitTorrent support was removed.
Release Notes: The filename format fix checks were updated so that they do not produce an error if that fix is not defined in your podgetrc. filename_formatfix9 was created to address issues with the Audioboo feed URL format. "?keyed=true&source=rss" was removed from the enclosure URL. String matches were made as generic as possible so that they might work for other feeds.
Release Notes: The default contents of the configuration file (podgetrc) was updated to further explain the SModcast updates.
Release Notes: A SModcast issue was fixed using a filename format fix that works in conjunction with filename_formatfix4. All but one of the filename format fixes were moved to a new function. This cleans up the main loop, making it easier to understand and maintain.
Release Notes: This release updates filename_formatfix to correct for feeds which place the name of the file to be downloaded in the parameter media_url=. It experiments with the order of filename format fixes. Those with more specific exclusionary conditions go first, and the more generic last.
Release Notes: This update addresses three issues. It has an update to filename_formatfix to correct for trailing characters on enclosures for the zdf.de podcasts. It has been fixed to display filenames while marking older podcasts as downloaded with the --recent option. Rather than display the current filename for each item, it would display the last modified filename repeatedly. filename_formatfix has been fixed to correct filenames downloaded from Vimcasts.org. Filename modification is now simpler and easier to understand.


text:
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Loews Philadelphia Hotel1200 Market StreetMeeting Rooms - Commonwealth A, B, C, DPhiladelphia, Pa.
You'll have an opportunity to discuss the latest in research, clinical trials, and clinical care with Pulmonary Hypertension experts. The event will be held in conjunction with the American Thoracic Society International Conference.
Paul R. Forfia, MDTemple University Medical Center
Brian D. Hanna, MDCM, PhDThe Children's Hospital of Philadelphia
Hear from experts in the pulmonary community and network with them afterwards. Presentations and speakers include:
**Come for the morning PH "Meet-the-Expert" Forum, enjoy a complimentary lunch and stay for the afternoon pulmonary sessions.
Hosted by:


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