Mitigation of isolation-associated adipocyte interleukin-6 secretion following rapid dissociation of adipose tissue.

Primary adipocyte isolation by collagenase digestion is a widely used technique to study metabolic regulation and insulin action in adipocytes. However, induction of a proinflammatory response characterized by enhanced secretion of interleukin (IL)-6 has been tightly linked to the isolation process itself. To test the hypothesis that the shaking mechanical force exerted on adipocytes stimulates inflammation during isolation, rat primary adipocytes were prepared by collagenase digestion in orbital shaking incubators maintained at varying speeds. Contrary to expectation, the isolation-induced release of IL-6 was attenuated by increasing the rotational speed of digestion and the concentration of collagenase, both of which resulted in rapid dissociation of adipocytes from the vasculature. In addition, the attenuation of IL-6 secretion was associated with decreased phosphorylation of the stress-related p38 mitogen-activated protein kinase (p38 MAPK) and preserved insulin action. The data suggest that optimization of parameters including, but not limited to, mincing technique, time of digestion, and collagenase concentration will make it possible to isolate primary adipocytes without activation of a proinflammatory response leading to elevated secretion of IL-6.

. Adipose tissue is composed of not only adipocytes but also a variety of immune cells and an elaborate stromal-vascular matrix ( 3,4 ). Considering the cellular heterogeneity of this tissue, dissecting out the contribution of different cell types to disease phenotypes is not a trivial endeavor. Isolating primary adipocytes from other adipose-resident cell types allows for the measurement of adipocyte-specifi c gene expression, metabolism, and responses to various hormones. The primary adipocyte isolation technique was pioneered in the 1960s by Martin Rodbell, who showed that a homogeneous preparation of adipocytes could be made by dissecting out the fat pads of rodents, cutting the adipose tissue into pieces, digesting it with a combination of collagenase and mechanical rotation, and subsequently removing the stromal-vascular cells via centrifugation ( 5 ).
Primary adipocyte isolation techniques based on Rodbell's original protocol have been widely applied, with a particular focus on insulin sensitivity as measured by levels of GLUT4 translocation, glucose and lipid metabolism, and more recently, adipokine secretion and action (6)(7)(8)(9)(10)(11)(12)(13). It is, therefore, important to understand how the isolation process itself and its constituent variables affect insulin sensitivity in liberated adipocytes.
Previous studies have demonstrated that the isolation process itself has negative effects on metrics of primary adipocyte infl ammation and insulin sensitivity. Ruan et al. showed that isolating adipocytes by collagenase digestion induced an acute and chronic release of the proinfl ammatory factors tumor necrosis factor-␣ (TNF-␣ ) and interleukin-6 (IL-6), respectively ( 14 ). Importantly, treatment with these cytokines has been shown to promote insulin resistance in 3T3-L1 adipocytes (15)(16)(17)(18)(19). Consistent with these fi ndings, Ruan et al. also showed that collagenase digestion of mouse epididymal adipose tissue triggered a downregulation of the expression of several genes involved in insulin Abstract Primary adipocyte isolation by collagenase digestion is a widely used technique to study metabolic regulation and insulin action in adipocytes. However, induction of a proinfl ammatory response characterized by enhanced secretion of interleukin (IL)-6 has been tightly linked to the isolation process itself. To test the hypothesis that the shaking mechanical force exerted on adipocytes stimulates infl ammation during isolation, rat primary adipocytes were prepared by collagenase digestion in orbital shaking incubators maintained at varying speeds. Contrary to expectation, the isolation-induced release of IL-6 was attenuated by increasing the rotational speed of digestion and the concentration of collagenase, both of which resulted in rapid dissociation of adipocytes from the vasculature. In addition, the attenuation of IL-6 secretion was associated with decreased phosphorylation of the stress-related p38 mitogen-activated protein kinase (p38 MAPK) and preserved insulin action. The data suggest that optimization of parameters including, but not limited to, mincing technique, time of digestion, and collagenase concentration will make it possible to isolate primary adipocytes without activation of a proinfl ammatory response leading to elevated secretion of IL-6. Supplementary key words obesity • infl ammation • digestion • collagenase and insulin sensitivity Dysregulation of adipose tissue has been shown to play a critical role in the etiologies of numerous pathologies, including obesity, insulin resistance, and atherosclerosis 10-15 ml of KRHB or RPMI 1640 media (Invitrogen). The cells were then poured through sterile gauze to remove any remaining clumps of nondissociated adipocytes or stromal-vascular components into graduated 15 ml polypropylene conical centrifuge tubes. Any remaining liquid below the layer of adipocytes was aspirated off using a pipetting needle (Popper and Sons). The volume of isolated adipocytes per gram of tissue digested was estimated using the printed graduations on the conical tubes. At this point, the isolated adipocytes were incubated further or lysed as described below.

Counting of cells released from adipose tissue during collagenase digestion
Fifty microliter aliquots of adipose tissue digestion mixtures (containing 3.3 mg collagenase/ml and rotating at 120 rpm at 37°C) were removed using wide bore tips at the specifi ed time points following the addition of the digestion solution. The digestion mixture was then spotted onto a 40 µm mesh cell strainer. Using a clean pipette tip, the cell suspension was drawn into the tip from the opposite side of the mesh to remove clumps of tissue. KRHB with 2% BSA was added to the cell suspensions to obtain cell densities suitable for the Countess automated cell counter (Invitrogen). Cell suspensions were then diluted 1:1 with a 0.4% Trypan Blue solution (Invitrogen). Ten microliters of these mixtures were added to Countess cell counting slides with 100 µm chamber depth prior to initiation of cell counting. Only live cells were counted.

Primary adipocyte size determination
One microliter of Nile red (AdipoRed, Lonza) was added to a 50 µL aliquot of isolated adipocytes in KRHB. Images of at least 45 cells from each group were taken from a minimum of six independent fi elds. Cells were visualized using a Leica DM IL inverted microscope equipped with a mercury arc lamp and an Omega XF100 fi lter set. Images were taken with a Qimaging CCD camera and QCapture imaging software. Cell diameters were measured using NIH ImageJ.

Insulin stimulation of primary adipocytes
Following the 22 h postdigestion incubation, isolated adipocytes were treated with or without 100 nM insulin (Humulin R, Eli Lilly) for 10 min at 37°C. Cells were then diluted 7-fold in KRHB and centrifuged for 3 min at 150 rcf. The buffer below the top adipocyte layer was aspirated off, and the pellet of cells was frozen at Ϫ 80°C until lysate preparation.

Cell lysate preparation and Western blotting
Adipocytes were isolated for lysate preparation either at the time of digestion completion or at the end of the 22 h postdigestion incubation following treatment with or without insulin. Homogenization buffer (5×; 25 mM HEPES pH 7.4, 125 mM NaCl, 10 mM sodium orthovanadate, 500 mM NaF, 10 µM microcystine LR, 1:10 Sigma protease inhibitor cocktails 1 and 2 supplemented with one Roche mini-Complete protease inhibitor cocktail without EDTA per 2 ml lysis buffer) was added to aliquots of isolated adipocytes at one-fi fth the volume of cells. The cells were then disrupted using a tissue homogenizer (Eberbach Con-Torque) with a Tefl on pestle rotating at medium-high speed signaling and sensitivity in adipocytes, including, insulin receptor substrate 2 (IRS2), phosphoinositide 3-kinase (PI3K), glucose transporter 4 (GLUT4), adiponectin, and peroxisome proliferator-activated receptor ␥ (PPAR-␥ ) ( 14 ). Interestingly, expression of genes involved in insulin responsiveness were downregulated even in adipocytes isolated from TNF-␣ ( Ϫ / Ϫ ) knockout mice, suggesting that increased IL-6, not TNF-␣ , secretion is the more likely cause of isolation-induced infl ammation and subsequent insulin desensitization ( 14 ). Data from humans also suggest a prominent role for IL-6 in obesity-induced adipocyte insulin resistance given that adipose tissue IL-6 content, but not TNF-␣ or leptin content, is strongly inversely correlated with insulin-stimulated glucose transport in isolated adipocytes ( 20 ).
The observation that the adipocyte isolation process leads to the downregulation of genes involved in insulin responsiveness while concomitantly inducing the release of the potent proinfl ammatory and insulin-desensitizing cytokine IL-6 underscores the need to reevaluate this procedure in order to mitigate this response to prepare the most physiologically relevant adipocyte populations ex vivo. Accordingly, the goal of this study was to determine conditions for the primary adipocyte isolation technique that mitigate the IL-6 release inherent to this procedure.

Animals
Retired male Sprague Dawley breeders ( ‫ف‬ 300 g) were obtained from Charles River Laboratories and maintained on a normal chow diet and a 12 h light-dark cycle. The Institutional Animal Care and Use Committee of the University of Arizona in accordance with the Public Health Service Animal Welfare Policy approved all experimental protocols.

Primary adipocyte isolation and quantifi cation of primary adipocyte yield
Following anesthesia by CO 2 inhalation and euthanasia by decapitation, approximately 2.0-3.0 g of epididymal fat were dissected out and placed in Krebs-Ringer-Henseleit buffer (KRHB, 30 mM HEPES, pH 7.4, 1 mM CaCl 2 , 120 mM NaCl, 4 mM KH 2 PO 4 , 1 mM MgSO 4 , 10 mM NaCO 3 , 200nM adenosine) containing 2% Fraction-V BSA (Sigma) prior to being weighed. Adipose tissue was then placed in digestion solution [KRHB containing 2% BSA and 3.3 mg/ml collagenase ( у 125 U/mg, Type II, Sigma) unless otherwise indicated] at a 1:1 ratio of milliliters of digestion solution to grams of adipose tissue. The tissue was then minced with surgical scissors for ‫ف‬ 2 min (at ‫ف‬ 3 openings and closings of blades per second), resulting in small ( ‫ف‬ 1-2 mm 3 ) pieces of adipose tissue. The digestion solution-tissue mixture was then transferred to a 250 ml siliconized-glass Erlenmeyer fl ask and incubated in orbital shaking incubators at 37°C, rotating at 30, 75, or 120 rpm. Digestions were deemed complete when no large clumps of tissue were visible and у 90% of the digestion solutiontissue mixture formed a uniform fi lm of cells when the mixture was tilted and allowed to run down the bottom of the fl ask. Upon digestion completion, the cells were poured into a siliconizedglass separating funnel for delicate washing and removal of stromal-vascular components without the need for centrifugation. Cells were washed four times in the separating funnel with 75 rpm 101 min ( P < 0.001) longer to reach digestion completion than tissue rotated at 120 rpm ( Fig. 2A ).
Consistent with the observation that slow dissociation at 30 rpm is associated with greater p38 MAPK activation, at the time of digestion completion, adipose tissue digested by rotating at 30 rpm secreted 7.3-and 11.1-fold ( P < 0.02) more IL-6 than adipose tissue digested by rotating at 75 or 120 rpm, respectively ( Fig. 2B ). Taking into consideration both the concentration of IL-6 at the time of digestion completion and the time to digestion completion, the rate of IL-6 secretion was not signifi cantly different when tissues were rotated at 30, 75, or 120 rpm ( Fig. 2C ).
To determine how rotational speed infl uences IL-6 production in adipocytes after cessation of the digestion procedure, isolated adipocytes were cultured for 22 h following for fi fteen 2 s strokes. Homogenates were then sonicated for 8 s at 30% amplitude (0.5 s ON and 0.5 s OFF) at 4°C, and then centrifuged at 14,000 rcf for 30 min at 4°C to separate the lipids from the supernatant, which was collected using a needle and syringe. Total protein concentrations were determined using the Bradford assay (Bio-Rad). Proteins were fractionated via SDS-PAGE and transferred to nitrocellulose paper. Immunoblotting was performed using purifi ed polyclonal rabbit antibodies against p38 mitogen-activated protein kinase (p38 MAPK), pan-Akt, and ␤ -actin (all from Cell Signaling) and purifi ed monoclonal mouse antibodies against phospho-p38 MAPK (Thr180/Tyr182) (Cell Signaling) and phospho-Akt (Thr308) (BD Biosciences). Secondary antibodies were IRDye 800 conjugated goat anti-mouse and IRDye 680 conjugated goat anti-rabbit antibodies (LI-COR). Imaging was performed using the Odyssey® Infrared Imaging System (LI-COR). To ensure equal protein loading, some blots were stained with 0.5% Ponceau S and then destained with ddH 2 O prior to blocking and immunoblotting.

ELISAs
Aliquots of the digestion solution were collected and fl ash frozen in liquid nitrogen at the time of digestion completion. Aliquots of the incubation media were collected and fl ash frozen in liquid nitrogen 22 h after the start of the incubation. The concentrations of IL-6 and IL-1 ␤ in the digestion solution and incubation media samples (adjusted for the cell:media volume) were determined using Quantikine rat IL-6 (R and D Systems) and rat IL-1 ␤ (Signosis) ELISA kits, respectively.

Statistical analysis
Data are presented as mean ± SE. Statistical comparisons between three groups were performed using one-way ANOVA with Tukey post hoc test ( Figs. 1B, 2A-E, 4C, 5A ). Statistical comparisons between two groups were performed using Student's paired two-tailed t -test ( Figs. 3A-C, 4B ) or one sample Student's two-tailed t -test ( Fig. 5C ). Groups not sharing a common letter are statistically different.

p38 MAPK activation and IL-6 secretion are attenuated in rapidly dissociated adipocytes
The traditional primary adipocyte isolation procedure employs both enzymatic and mechanical digestion of adipose tissue. Previous studies have shown that shearing mechanical stress induces p38 MAPK phosphorylation, which subsequently leads to increased IL-6 production ( 21 ). Therefore, we hypothesized that delicate digestion at a slow rotational speed would reduce shearing mechanical stress as measured by p38 MAPK phosphorylation and thereby reduce IL-6 secretion.
Contrary to our expectations, adipocytes isolated by rotating ‫ف‬ 2.0-3.0 g of adipose tissue at 30 rpm had ‫ف‬ 2.7-fold ( P < 0.007) greater p38 MAPK phosphorylation than adipocytes isolated by rotating at 75 or 120 rpm ( Fig. 1A, B ). As expected, increasing the rotational speed and, therefore, mechanical digestion, reduced the time to digestion completion. On average, it took tissue rotated at 30 rpm 154 min ( P < 0.0001) and 255 min ( P < 0.0001) longer to reach digestion completion than tissue rotated at 75 and 120 rpm, respectively. On average, it took tissue rotated at densitometry quantifying the ratio of phospho-p38 MAPK (Thr180/Ty182) to total p38 MAPK intensity at the time of digestion completion in adipocytes isolated from tissue digested at a rotational speed of 30, 75, or 120 rpm. The relative amount of total protein loaded in the different lanes was assessed by staining with Ponceau S prior to blocking and immunoblotting (n = 4 per group). Data were normalized to phospho:total p38 MAPK ratio at 30 rpm. LC/MS/MS was performed on the predominant band running at ‫ف‬ 35 kDa in the Ponceau S stain. Proteins identifi ed by Scaffold 3 software as being present with greater than 99.9% probability included carbonic anhydrase 3, 14-3-3 protein ␥ , electron transfer fl avoprotein subunit ␤ , phosphoglycerate mutase 1, 14-3-3 protein ␤ /alpha, and adiponectin. One-way ANOVA with Tukey's post hoc test was used for all between-group analyses. Groups not sharing a common letter are signifi cantly different ( P < 0.007).
variation regarding technical details, including the mincing technique, collagenase concentration, time to digestion completion, rotational shaking speed, digestion container, digestion buffer, the ratio of tissue to volume of digestion buffer, and the amount of tissue digested ( Table 1 ). Many of these studies used a lower collagenase concentration (0.5-1.0 mg/ml) than that used in the studies presented here or in Rodbell's initial studies (3.3 mg/ml). Given that a lower collagenase concentration is expected to decrease the rate of enzymatic digestion and our observation that a more rapid dissociation of adipocytes leads to attenuated IL-6 secretion, we directly compared the time to digestion completion and IL-6 secretion between tissues digested with 1.0 versus 3.3 mg collagenase/ml in a subset of our experiments.
To determine the effects of collagenase concentration on the time to digestion completion and IL-6 secretion independent of rotational speed, paired observations from an equal number of experiments with rotation at 30, 75, and 120 rpm were pooled. These experiments showed that adipocytes isolated with 1.0 mg collagenase/ml secreted 1.8-fold ( P < 0.02) more IL-6 at 22 h post digestion completion than adipocytes isolated with 3.3 mg collagenase/ml ( Fig. 3A ). This was accompanied by a 54 min ( P < 0.03) increase in the amount of time for digestion to reach completion ( Fig. 3B ) and a tendency for increased IL-6 concentrations at the time of digestion completion (10.8-fold increase, P = 0.1044, Fig. 3C ) when adipose tissue was digested with 1.0 versus 3.3 mg collagenase/ml. Interestingly, while increases in the time to digestion completion and IL-6 secreted after overnight culture were observed in washing and removal of the stromal-vascular components. Adipocytes isolated by rotating at 30 rpm secreted 1.6-and 7.0-fold ( P < 0.04) more IL-6 than adipocytes isolated by rotating at 75 or 120 rpm, respectively ( Fig. 2D ). Importantly, the concentrations of IL-6 secreted from adipocytes isolated by rotating at 30 and 75 rpm ( ‫ف‬ 2,500 pg/ml and ‫ف‬ 1,500 pg/ml, respectively) approach the concentrations reported to reduce insulin sensitivity in adipocytes (5,000-20,000 pg/ml) ( 16,19 ).
Interleukin-1 ␤ (IL-1 ␤ ), like IL-6, has been shown to reduce insulin sensitivity in adipocytes ( 22,23 ). As insulin sensitivity is a common metric assessed in isolated primary adipocytes, we sought to measure the secretion of IL-1 ␤ in response to various rotational speeds of digestion. At the time of digestion completion, the absolute concentrations of IL-1 ␤ at all three rotational speeds were below the assay's limit of detection (data not shown). Twenty-two hours post digestion completion, IL-1 ␤ secretion showed a strong tendency to be lower in adipocytes isolated by rotating at 120 rpm compared with adipocytes isolated by rotating at 30 or 75 rpm ( P = 0.0507, Fig. 2E ). The highest concentration of IL-1 ␤ secreted by isolated adipocytes in these experiments ( р 23 pg/ml), however, was much lower than the concentration of IL-1 ␤ reported to reduce insulin sensitivity in adipocytes (20,000 pg/ml) ( 22 ).

High collagenase concentration during digestion is associated with reduced IL-6 secretion in dissociated adipocytes
A search of the literature for studies employing a primary adipocyte isolation procedure revealed substantial insulin-stimulated Akt phosphorylation. There was no difference in the average diameter of adipocytes isolated with 1.0 versus 3.3 mg collagenase/ml ( Fig. 4A, B ). In addition, there was no difference in the average diameter or the distribution of diameters of adipocytes isolated using 3.3 mg collagenase/ml with rotation at 30, 75, or 120 rpm ( Fig. 4C, D ). There was no difference in adipocyte yield when adipocytes were isolated using 3.3 mg collagenase/ml with rotation at 30, 75, or 120 rpm ( Fig. 5A ). Lastly, insulin-stimulated Akt phosphorylation at 22 h post digestion completion was highest in adipocytes isolated using 3.3 mg collagenase/ml with rotation at 120 rpm ( P < 0.04) ( Fig. 5B, C ). Surprisingly, there was essentially no insulin-stimulated Akt phosphorylation observed in adipocytes isolated using 3.3 mg collagenase/ml with rotation at 30 and 75 rpm.

DISCUSSION
Metabolic studies of isolated primary adipocytes have become widespread as the need to determine the mechanisms of metabolic diseases, including obesity and diabetes, has risen. The primary adipocyte isolation procedure groups treated with 1.0 versus 3.3 mg collagenase/ml, regardless of rotational speed, adipocytes prepared by rotating at 120 rpm secreted 2.4-fold more IL-6 after overnight culture, even though they only spent on average 9 additional minutes (23-32 min) in the collagenase digestion mixture ( Fig. 3D ). These data suggest that the prolongation of the time to reach digestion completion due to lowering the collagenase concentration could be responsible for the increased IL-6 secretion at 22 h post digestion completion, but it is likely not the only factor.
Importantly, upon quantifying the number of cells liberated at specifi c times during digestion at 120 rpm using 3.3 mg collagenase/ml we found that greater than 50% of the cells were liberated after just 2.5 min and 100% of the cells were liberated after 10-15 min ( Fig. 3E ).

Effects of collagenase concentration and rotational speed on adipocyte integrity
Both rotation speed and duration of digestion could have a negative impact on the physical and functional integrity of isolated adipocytes. Therefore, the following metrics of integrity were assessed in adipocytes isolated under the various conditions: i ) cell diameter, ii ) adipocyte yield, and iii ) associated with p38 MAPK activation. For tissues weighing ‫ف‬ 2.0-3.0 g, digesting at a high rotational speed (75 or 120 rpm) resulted in lower p38 MAPK phosphorylation compared with rotation at 30 rpm ( Fig. 1 ). Consistent with the observation that p38 MAPK activation induces IL-6 production ( 21 ), digestion at these high rotational speeds also resulted in lower IL-6 secretion at the time of digestion completion and 22 h post digestion completion compared with rotating at 30 rpm. In addition, the reduction in IL-6 secretion at both of these time points was correlated with a faster time to digestion completion ( Fig. 2 ).
The rate of IL-6 secretion during the digestion process was comparable for tissues rotated at 30, 75, and 120 rpm ( Fig. 2C ). Therefore, because it took tissues rotated at 30 rpm longer to reach digestion completion than tissues rotated at 75 rpm, which took longer than tissues rotated at 120 rpm ( Fig. 2A ), the hierarchy of the absolute yield of IL-6 at the time of digestion completion was 30 > 75 > 120 rpm ( Fig. 2B ). Given that adipocytes isolated by rotating at 30, 75, and 120rpm were incubated for the same amount of time during the post-digestion experiments (22 h), the differences in IL-6 concentrations 22 h post digestion completion ( Fig. 2D ) must have been due to differences in the rate of IL-6 secretion between adipocytes isolated at the various rotational speeds. The rate of IL-6 secretion during the 22 h incubation may have been infl uenced by used in these various studies varies little with regard to the primary steps of i ) tissue dissection and mincing, ii ) collagenase digestion, and iii ) removal of stromal-vascular components. However, technical details of the procedure, including the mincing technique, collagenase concentration, time to digestion completion, rotational shaking speed, digestion container, digestion buffer, the ratio of tissue to volume of digestion buffer, and the amount of tissue digested appear to vary considerably among studies ( Table 1 ). Many of these details are often not recorded, presumably because they are not considered important.
Previous work by Ruan et al. has shown that the primary adipocyte isolation process itself induces an infl ammatory response as measured by secretion of TNF-␣ and IL-6, which have been shown to have insulin-desensitizing effects (14)(15)(16)(17)(18)(19)24 ). This prior study additionally demonstrated that the isolation process leads to the downregulation of several genes involved in insulin signaling. Considering that insulin sensitivity is a commonly assessed metric in primary adipocyte studies, these discoveries highlighted the need to minimize the dysregulation of liberated adipocytes to ensure that experimental outcomes are physiologically relevant.
Here we show that isolation-induced infl ammation in primary adipocytes, as measured by IL-6 secretion, appears to arise primarily from the length of digestion time and is at 120 rpm in 1.0 mg collagenase/ml, just a small (9 min) increase in the absolute amount of time for digestion to reach completion resulted in a disproportionate increase in IL-6 secretion after overnight culture compared with digestion in 3.3 mg collagenase/ml ( Fig. 3D ). This likely refl ects the inherent lack of precision in assessing the completeness of digestion by visual inspection. At the high rotational speed of 120 rpm, mechanical agitation may have caused most of the adipocytes to become separated from each other, leading to the appearance of digestion completion by visual inspection. However, the relative lack of extracellular matrix breakdown in low collagenase concentration could allow some macrophages to remain attached to single adipocytes, leading to increased secretion of IL-6 in overnight cultures. Future experiments should examine the relationship between collagenase concentration and the presence of macrophage-specifi c markers in adipocyte preparations.
Adipocyte integrity, as measured by adipocyte size and yield, were comparable between tissues digested with 3.3 mg collagenase/ml at 30, 75, and 120 rpm ( Fig. 4C, D and Fig. 5A ). Importantly, however, adipocytes isolated with rotation at 120 rpm exhibited the highest insulin sensitivity 22 h post digestion completion ( Fig. 5B, C ). The absence of insulin-stimulated Akt phosphorylation in adipocytes isolated with rotation at 30 or 75 rpm was unexpected. One possible explanation is that the commercial BSA stock was contaminated with some insulin. Therefore, the groups not treated with insulin may not refl ect true basal, noninsulin-stimulated conditions. Despite this possibility, the insulin stimulation assay conditions were identical for all adipocytes isolated at the various rotational speeds and the adipocytes isolated with rotation at 120 rpm did indeed exhibit a ‫ف‬ 2-fold increase in Akt phosphorylation upon insulin stimulation.
The very short time (27 min) that it took adipose tissue digestions rotated at 120 rpm to reach digestion completion was likely due to three primary factors: i ) the mincing technique, ii ) the high concentration of collagenase (3.3 mg/ml), and iii ) the high rotational speed. The mincing technique employed in the primary adipocyte isolation procedure described herein involved the mincing of adipose tissue bathed in the digestion solution for ‫ف‬ 2 min with surgical scissors, resulting in small ( ‫ف‬ 1-2 mm 3 ) pieces of adipose tissue. This mincing technique greatly increases the surface area of the adipose tissue, thereby increasing the rate of both enzymatic and mechanical digestion. Given the strong impact of the mincing technique on the rate of digestion, this step should be standardized in future primary adipocyte isolation protocols.
As previously alluded to, the benefi t of decreasing the time to adipose tissue digestion completion could be due to infl uences from cells other than adipocytes within the adipose tissue. It has been previously shown that >90% of the proinfl ammatory cytokines produced by the adipose tissue originates from nonfat cell types ( 26 ). It is possible that the cells within the adipose tissue stromal-vascular matrix and resident immune cells produce the majority of proinfl ammatory cytokines in response to the stress of the the rotational speed of digestion, the time to reach digestion completion, or the absolute amount of IL-6 produced during the digestion. Interestingly, chronic IL-6 treatment of differentiating 3T3-F442A adipocytes has been shown to increase endogenous IL-6 secretion in the resultant mature adipocytes ( 25 ).
Consistent with the observed positive correlation between time to digestion completion and IL-6 secretion in our initial studies, when a lower concentration of collagenase (1.0 mg/ml) was used, digestion at all three rotational speeds took longer to reach digestion completion and resulted in higher IL-6 secretion 22 h post digestion completion compared with digestions using a higher concentration of collagenase (3.3 mg/ml, Fig. 3A-C ). However, the length of time to reach digestion completion is likely not the only factor infl uencing IL-6 secretion. For adipocytes digested One-way ANOVA with Tukey's post hoc test was used for all between-group analyses. (B) Representative Western blot and (C) densitometry quantifying the ratio of phospho-Akt (Thr308) to total-Akt 22 h post digestion completion in adipocytes isolated from tissues digested at rotational speeds of 30, 75, or 120 rpm with 3.3 mg collagenase/ml in the absence or presence of 100 nM insulin for 10 min at 37°C (n = 4 per rotational speed per treatment). The relative amount of total protein loaded in the different lanes was assessed by immunoblotting for ␤ -actin. Data were normalized to "no insulin" at each rotational speed for each experiment. For each rotational speed, a one sample Student two-tailed t -test with hypothetical mean set to 1 for groups not treated with insulin was used to compare phospho:total Akt ratio in the absence or presence of insulin. Groups not sharing a common letter are signifi cantly different ( P < 0.04).
vessel, orbit size of the orbital shakers, and mass of the digestion mixture, could affect the rate of adipocyte dissociation. Indeed, we observed that shaking larger adipose tissue samples of ‫ف‬ 5.0-6.5 g in 3.3 mg collagenase/ml at 75 rpm led to a faster time to digestion completion and lower adipocyte IL-6 secretion after 22 h in culture compared with shaking tissues weighing ‫ف‬ 2.0-3.0 g (data not shown). However, when both rotational speed and digestion mixture mass are suffi ciently high, the centrifugal force may become too large and cross a mechanical stress threshold beyond which adipocytes may not tolerate well. Consistent with this hypothesis, digesting ‫ف‬ 5.0-6.5 g of adipose tissue at 120 rpm led to increased adipocyte IL-6 secretion 22 h post digestion completion compared with digesting ‫ف‬ 2.0-3.0 g of tissue (data not shown). These observations suggest that there may be a fi ne balance between rapidity of digestion and induction of stress via mechanical force. digestion process. Readily removing these cells from the proximity of adipocytes using conditions for rapid dissociation may diminish their ability to induce an infl ammatory response in adipocytes in a paracrine manner. In addition, rapid digestion of adipose tissue may reduce isolationinduced infl ammation by shortening the time in which the inner region of the tissue pieces are exposed to a hypoxic environment, which has been shown to induce IL-6 expression ( 27 ).
The observation that rotational speed and duration of digestion are likely not the only factors infl uencing IL-6 secretion underscores the need to evaluate multiple factors in the adipocyte isolation process. For example, the degree of mechanical force exerted on the adipose tissue likely contributes to the rate of digestion. Consideration of the centrifugal force equation (Fc = mv 2 /r, where Fc = centrifugal force, m = mass, v = velocity, r = radius) suggests that parameters, such as the radius of the digestion Taken together, the evidence presented here suggests that the proinfl ammatory IL-6 secretion associated with the primary adipocyte isolation procedure can be mitigated if adipocytes are rapidly dissociated. Without sacrifi cing yield or adipocyte insulin sensitivity, this rapid dissociation can be achieved by thoroughly mincing the adipose tissue, using a high concentration of collagenase (3.3 mg/ml), and optimizing the speed of rotation to deliver an appropriately high degree of mechanical force (120 rpm for ‫ف‬ 2.0-3.0 g of adipose tissue). Under these conditions, the duration of the digestion can be modifi ed depending on the yield required ( ‫ف‬ 100% of cells are liberated after 15 min). These suggested guidelines will hopefully enhance the reproducibility and physiological relevance of future primary adipocyte studies.