Learning Objectives

We measured the basal and stimulated lipolytic rate of in vitro differentiated adipocytes. Primary preadipocytes from inguinal white adipose tissue were differentiated into adipocytes by the treatment of confluent cells with 5 µM dexamethasone, 0.5 mM IBMX, 1 µg/mL insulin, and 1 µM troglitazone for 4 days, followed by an additional 3 day treatment with 1 µg/mL insulin. Cells were incubated in media without insulin for 24 h prior to the lipolysis assay. At time = 0h, the cells were washed once with PBS, then phenol red-free DMEM with 2% BSA, containing either 10 µM CL-316,243 or vehicle control, was added to each well of the 12-well plate. At time = 1 h, 2 h, 3 h, and 4 h, 50% of the media was collected and replaced with 2% BSA in phenol red-free DMEM. FFA and glycerol levels were measured in the collected media and the moles of FFA, and glycerol secreted into media was calculated at each time point (Figure 2A,B). FFA and glycerol production were linear for the 4 h assay, with R² values of 0.98 and above. The 4 h FFA levels were slightly low, indicating that lipolytic rates may have been slowing; however, analysis with and without the 4 h time point did not have a significant impact on the calculated lipolytic rate. Stimulated lipolytic rates were significantly higher than basal rates (Figure 2C). Lipolytic stimulation resulted in the production of FFA and glycerol at a near 3:1 molar ratio in all collected samples, as would be expected from complete triglyceride lipolysis without significant reuptake or retention (Figure 2D). However, in the unstimulated cells, the ratio was closer to 1, suggesting a non-lipolytic source of glycerol^48,49,50.

Figure 2: Lipolytic rate in in vitro differentiated primary adipocytes. Preadipocytes were differentiated in a 12-well plate. Insulin was removed from the media 24 h prior to the lipolytic assay. At time = 0 h, the cells were stimulated with 10 µM CL-316,243 (CL) or treated with vehicle (V) control media. The nmol of (A) FFA and (B) glycerol produced in each well by each time point were plotted over time, and a linear regression line was fitted for each individual well. (C) Rate of FFA production. (D) Rate of glycerol production. Data are represented as mean ± SEM. (E) Molar ratio of FFA:glycerol in the collected media at each time point. Statistical analysis in (C) and (D) was performed using a student's t-test; the effect of CL was significant at α = 0.05. Please click here to view a larger version of this figure.

In cell culture, the monolayer of cells is in direct contact with the media, while in tissues cultured ex vivo, cells in the center are not in contact with the media. Thus, in ex vivo cultures, FFAs are more likely to be retained within the tissue. Larger chunks of tissue, which have a lower surface area to volume ratio, exhibit higher rates of FFA retention, resulting in lower FFA:glycerol molar ratios (Figure 3A). With decreasing tissue chunk size, the molar ratio of FFA:glycerol released approaches 3:1 (Figure 3A). However, tissue chunks can also be too small. In addition to the challenge of working with tiny pieces of adipose tissue, 1-2 mg chunks of adipose tissue exhibit reduced lipolytic rates, suggesting a reduced viability and functionality of the tissue (Figure 3B,C). While it is tedious and time consuming to cut adipose tissue into chunks of consistent size and shape, it is required to obtain comparable and reliable results. We recommend 5 to 10 mg chunks of adipose tissue, but consistency is most important. Lipolytic rates can still be reproducibly measured in larger tissue chunks, however, it is important to consider the feedback inhibition of lipolysis by FFAs^{63,64,65,66,67}.

Figure 3: Effect of tissue chunk size on FFA release and lipolytic rate. Gonadal white adipose tissue from a high fat diet fed female C57Bl/6J mouse was collected and cut into chunks of varying size. All wells contained 25-30 mg of adipose tissue in total; for the 25 mg wells, this was one chunk of tissue; 10 mg wells had three chunks of tissue each of ~ 10 mg, the 5 mg wells contained five or six chunks of tissue, and the 2 mg wells contained 13-16 chunks. At time = 0 h, lipolysis was stimulated with 0.5 µM CL-316,243 (CL), or the control wells were treated with vehicle (V). The calculated lipolytic rate from samples collected after 1 h, 2 h, and 3 h is plotted for (A) FFAs and (B) glycerol. (C) Molar ratio of FFA:glycerol production in each well. Data are represented as mean ± SEM. Statistical analysis in (A) and (B) was performed using a two-way ANOVA with a Holm-Sidak post-hoc analysis, α = 0.05. The effect of CL was significant in all samples. Within the CL treated samples, the rates of FFA and glycerol production were significantly different in all pair-wise comparisons, except the 10 mg versus 5 mg samples. Please click here to view a larger version of this figure.

The negative impact of FFA retention can be observed in the 25 mg tissue chunks, which exhibit lower rates of both FFA and glycerol production upon stimulation (Figure 3B,C). This feedback inhibition is also apparent when the FFA binding capacity of the media is too low (i.e., there is not enough BSA in the media). When the BSA in the media was reduced to 0.5%, FFA release was reduced more than fourfold, and glycerol release reduced nearly twofold (Figure 4A,B). As an easy rule of thumb, micromolar levels of FFA in the media should not approach the percentage of BSA in the media (i.e., FFA levels should remain below 5 mM FFA in 5% BSA media and 0.5 mM in 0.5% BSA media [at this level there is 20:3 molar ratio of FFA:BSA]). To test the maximal BSA buffering capacity of the preparation of BSA media, collect media from a 24 h stimulated sample, and measure the concentration of FFA (the concentration will be high, so a lower sample volume or sample dilution is recommended). The FFA accumulation in the 5% BSA media after 24 h stimulation was about 5 mM, while the FFA content of the 0.5% BSA media was only 0.6 mM (Figure 4C). Looking at the FFA concentration in the collected media, it can be seen that the 0.5% BSA media FFA binding capacity is overburdened (Figure 4D). While FFA levels in the 5% BSA media are much higher, they do not exceed the buffering capacity of the media (Figure 4D). Ideally, the FFA concentration in the media should remain below a 3:1 FFA:BSA molar ratio (i.e., 2.3 mM FFA in 5% [0.75 mM] BSA).

Figure 4: Insufficient BSA levels result in reduced apparent lipolytic rates. Gonadal white adipose tissue from a high fat diet fed female C57Bl/6J mouse was collected and cut into ~5 mg chunks. A total of 20-30 mg of adipose tissue was placed in each well. At time = 0 h, the lipolysis was stimulated with 0.5 µM CL-316,243 (CL), or the control wells were treated with vehicle (V) in either 5% BSA media or media containing only 0.5% BSA. The calculated lipolytic rate from the samples collected after 4 h is plotted for (A) FFAs and (B) glycerol. The effect of CL was significant in all samples. Within the CL-treated samples, the rates of both FFA and glycerol production were significantly different between the 5% and 0.5% BSA media conditions. (C) FFA levels in the media from the stimulated wells after 24 h. (D) FFA levels in the samples collected at 4 h. (E) FFA standard curves with and without various preparations of 5% BSA in DMEM. Optical density calculated as (Reading B: A₅₅₀ – A₆₆₀) – (Reading A: A₅₅₀ – A₆₆₀). (F) Glycerol standard curve with and without BSA. Optical density is absorbant at 540 nm (A₅₄₀). OD values at 5.6 mM were not linear and thus not included in the standard curve. (G) Rate of FFA release from female gonadal adipose tissue using different types of BSA in the assay media. Data are represented as mean ± SEM. Statistical analysis in (A) and (B) was performed using a two-way ANOVA with a Holm-Sidak post hoc analysis, * p value < 0.05. Please click here to view a larger version of this figure.

It is important to point out that some preparations of BSA contain FFAs. Each lot of BSA should be tested to ensure that the FFA content is negligible (note that some BSA preparations are marketed as FFA free). While not specifically marketed as FFA-free, the BSA used here does not contain detectable FFAs. We tested the assay media containing FFA-free BSA (Equitech Bio Inc, BAH66), the BSA used in experiments here (Sigma, A9418), and a cruder (less expensive) fraction V BSA (Sigma, 810531). The FFA-free BSA and A9418 BSA both did not produce any background signal or change the standard curve slope or intercept; the standard curves were superimposable (Figure 4E). No impact of the BSA DMEM was observed on the glycerol standard curve either (Figure 4F). The fraction V BSA, on the other hand, did produce a background signal in the FFA assay, which equated to an FFA concentration of 0.3 mM (Figure 4E). This background shifted the standard curve up, but did not significantly impact the slope, indicating that background subtraction is sufficient. We also performed a stimulated lipolysis experiment using these three different types of BSA, and found that after background subtraction, the calculated rate of lipolysis did not differ between BSA formulations (Figure 4F). However, this is often not the case, especially with less pure BSA preparations which can easily contain insulin or other components that impact the rate of lipolysis. Each lot of BSA should be tested and validated, and used consistently (i.e., samples assayed with different lots of BSA are not comparable). A full standard curve with the addition of BSA and DMEM should be performed when validating each lot of BSA to ensure that it does not contain anything that interferes with the enzymes in the colorimetric assays.

Taking serial samples and replacing the volume with fresh media helps to lower the FFA load in the media throughout the experiment. We measured lipolytic rates in the gonadal adipose tissue from a female mouse fed a high fat diet for 4 months. In this experiment, 5-8 mg tissue chunks (total weight of 25-30 mg) were incubated in 200 µL of 5% BSA media, and 100 µL was collected and replaced at 1 h, 2 h, 3 h, and 4 h. FFA and glycerol levels in the collected media were measured. At 3 h, the FFA levels in the media had reached the danger zone of 2.3 mM (Figure 5A). Feedback inhibition of lipolysis was suggested by the lack of increase in media FFA and glycerol levels at 4 h (Figure 5A,B). After calculating the µmol of FFA and glycerol released at each time point and fitting a linear curve, it is apparent that the lipolytic rate starts to decline at 4 h in the stimulated samples, with R² values as low as 0.976 for FFA and 0.983 for glycerol (Figure 5C,D). Looking at the residual plot, it is clear that the 4 h values are below the linear trend (i.e., the residuals are negative) (Figure 5E,F). Exclusion of the 4 h time point increased the R² values to 0.997 and 0.998 and above for FFA and glycerol, respectively. Inclusion of the 4 h time point causes a small but significant decrease in the calculated FFA and glycerol release rates (Figure 5G,H).

Figure 5: Calculation of linear lipolytic rate in ex vivo adipose tissue. Gonadal white adipose tissue from a high fat diet fed female C57Bl/6J mouse was collected and cut into ~5 mg chunks. A total of 20-30 mg of adipose tissue was placed in each well. At time = 0 h, the lipolysis was stimulated with 0.5 µM CL-316,243 (CL), or the control wells were treated with vehicle (V). Media collections were performed at 1 h, 2 h, 3 h, and 4 h. (A) FFA and (B) glycerol levels in the media. (C) FFA and (D) glycerol production plotted over time. Residual plates for (E) FFA and (F) glycerol production over time. Rate of release of (G) FFA and (H) glycerol, calculated with and without the 4 h time point. (I) FFA release in female gonadal adipose tissue. Nearly 50% of media changed at 1 h, 2 h, and 3 h. Between hours, the 15 min time points collected 2.75% of the media without replacement. Data are represented as mean ± SEM. Statistical analysis in (G) and (H) was performed using a two-way ANOVA with a Holm-Sidak post-hoc analysis, * p value < 0.05. The effect of CL was significant in all samples (p value < 0.05). Within the CL treated samples, the rates of both FFA and glycerol production were significantly different between calculations with and without the 4 h time point. Please click here to view a larger version of this figure.

Unlike previous protocols, this protocol utilizes multiple time points to determine the lipolytic rate, thereby reducing measurement error and providing internal validation that the linear rate of lipolysis is being measured. To maintain lipolysis in the linear phase at each collection point, 50% of the media is collected and replaced. Since linearity is critical, we wanted to ensure that the addition of fresh media was not causing a burst of lipolysis at each addition. To validate the linearity between time points, we took a very small sample (2.75%) every 15 min between the hourly time points from 1 h to 3 h, without replacement. At 1 h, 2 h, and 3 h, the normal 50% sample collection was made and replaced. The rate of FFA release was linear between the usual time points, indicating that there was not a burst of lipolysis with each addition of fresh media (Figure 5I).