Optimized conditions for measuring lipolysis in murine primary adipocytes

The current literature on lipolysis in murine primary adipocytes is rife with experiments performed under conditions not optimized for reproducible and reliable results. Here, we present conditions for optimizing the measurement of lipolysis in murine adipocytes. We demonstrate that adenosine management is of paramount importance in evaluating the lipolytic response under basal and stimulated conditions. Also, adipocyte concentrations in the 10,000–15,000 cells per milliliter range produce a greater increase in stimulated lipolysis than higher concentrations, and the response is further enhanced by agitating the cells. The current literature on lipolysis in murine primary adipocytes is rife with experiments performed under conditions not optimized for reproducible and reliable results. Here, we present conditions for optimizing the measurement of lipolysis in murine adipocytes. We demonstrate that adenosine management is of paramount importance in evaluating the lipolytic response under basal and stimulated conditions. Also, adipocyte concentrations in the 10,000–15,000 cells per milliliter range produce a greater increase in stimulated lipolysis than higher concentrations, and the response is further enhanced by agitating the cells. Nearly 20 years ago, we embarked on a series of experiments to explore the basis for the high variability among published studies on lipolysis in isolated adipocytes. At that time, the vast majority of such studies were performed with rat adipocytes, isolated according to the classical method of Rodbell (1Rodbell M. Adipocyte isolation.J. Biol. Chem. 1964; 239: 375-380Abstract Full Text PDF PubMed Google Scholar). We published several papers on the handling and manipulation of isolated adipocytes (2Londos C. Honnor R.C. Dhillon G.S. cAMP-dependent protein kinase and lipolysis in rat adipocytes. III. Multiple modes of insulin regulation of lipolysis and regulation of insulin responses by adenylate cyclase regulators.J. Biol. Chem. 1985; 260: 15139-15145Abstract Full Text PDF PubMed Google Scholar, 3Honnor R.C. Dhillon G.S. Londos C. cAMP-dependent protein kinase and lipolysis in rat adipocytes. II. Definition of steady-state relationship with lipolytic and antilipolytic modulators.J. Biol. Chem. 1985; 260: 15130-15138Abstract Full Text PDF PubMed Google Scholar, 4Honnor R.C. Dhillon G.S. Londos C. cAMP-dependent protein kinase and lipolysis in rat adipocytes. I. Cell preparation, manipulation, and predictability in behavior.J. Biol. Chem. 1985; 260: 15122-15129Abstract Full Text PDF PubMed Google Scholar) required to provide optimal and reproducible results in the measurement of lipolysis, because most published studies revealed a high level of variability in results. In recent years, an increasing number of papers have appeared on the behavior of isolated murine adipocytes, and once again, the literature reveals a high level of variability in the magnitude of stimulation achieved with β-adrenergic receptor agonists or with other stimulants that increase cAMP concentrations and PKA activity. In our earlier studies, several factors emerged as important for the reproducible assay of adipocyte lipolysis, not the least of which was careful control of the ambient adenosine concentration (4Honnor R.C. Dhillon G.S. Londos C. cAMP-dependent protein kinase and lipolysis in rat adipocytes. I. Cell preparation, manipulation, and predictability in behavior.J. Biol. Chem. 1985; 260: 15122-15129Abstract Full Text PDF PubMed Google Scholar). Adipocytes contain a high-affinity A1 adenosine receptor that is linked to Gi, the adenylyl cyclase inhibitory G protein. For two reasons, it is important to control the adenosine concentration. First, in the absence of adenosine, adipocytes may exhibit constitutively high lipolytic activity. Second, based on various factors, such as the season of the year and immediate dietary history, basal activity may be relatively high, thus blunting the magnitude of stimulation over basal that may be achieved with a lipolytic stimulant (i.e., signal-to-noise ratio). Another important feature is the control of fatty acids that are released upon stimulation of lipolysis. Fatty acids are strongly inhibitory toward the receptor-mediated activation of adenylyl cyclase (5Fain J.N. Shephard R.E. Inhibition of adenosine 3′:k′-monophosphate accumulation white fat acids, lactate, and beta-hydroxybutyrate.J. Lipid Res. 1976; 17: 377-385Abstract Full Text PDF PubMed Google Scholar). Appropriately low fatty acid concentrations are achieved using low adipocyte concentrations, such that the ambient BSA in the assay medium is sufficient to bind all free fatty acids. Likewise, adequate mixing of the incubation medium is also essential to provide access of free fatty acids to the medium BSA. Otherwise, the adipocytes rise to the top of the incubation tube and present excess fatty acids to adjacent adipocytes in the upper layer, thus inhibiting adenylyl cyclase activity. Here, we present conditions for obtaining reproducible and optimal results for measuring lipolysis in primary murine adipocytes. All reagents were obtained from Sigma-Aldrich (St. Louis, MO) and were prepared and manipulated in an Adipocyte Incubation Solution (AIS) unless indicated otherwise. The AIS contained Krebs Ringer Bicarbonate HEPES buffer (containing 10 mM bicarbonate and 30 mM HEPES, pH 7.4) supplemented with 3% (w/v) fatty acid free bovine albumin fraction V (number 820025; ICN Biomedical, Inc.). Where indicated, the adenosine deaminase-resistant (ADA) A1 receptor agonist, (−)-N6-(2-phenyl-isopropyl)-adenosine (PIA), was present at 100 nM, (−)-isoproterenol (ISO) was present at 10 μM, and ADA was present at 1 U/ml. It is important to use enzyme stock supplied in ammonium sulfate and not glycerol suspensions, because the glycerol content interferes with the glycerol assay used in these studies. The cells were isolated in AIS fortified with 500 nM adenosine and 3 mg/ml type 1 collagenase (Worthington Biomedical Corp., Lakewood, NJ). The optimal concentration of adenosine used for adipocyte isolation and washes was based on the finding that mouse adipocytes were ∼10-fold less sensitive compared with rat adipocytes (J. Tansey and C. Londos, unpublished observation). Because of lot-to-lot variation in collagenase activity, the optimal concentration of collagenase may range from 1 to 3 mg/ml; we determined that 3 mg/ml collagenase stock was optimal for the digestion of adipose tissue samples in this study. Procedures involving animals were carried out in accordance with the guidelines specified by the National Institute of Diabetes and Digestive and Kidney Diseases Animal Care and Use Committee. Adipocytes were isolated according to the classical Rodbell method (1Rodbell M. Adipocyte isolation.J. Biol. Chem. 1964; 239: 375-380Abstract Full Text PDF PubMed Google Scholar) with modifications, such as the inclusion of adenosine in the medium during cell isolation to suppress lipolysis, as recommended by Honnor, Dhillon, and Londos (3Honnor R.C. Dhillon G.S. Londos C. cAMP-dependent protein kinase and lipolysis in rat adipocytes. II. Definition of steady-state relationship with lipolytic and antilipolytic modulators.J. Biol. Chem. 1985; 260: 15130-15138Abstract Full Text PDF PubMed Google Scholar, 4Honnor R.C. Dhillon G.S. Londos C. cAMP-dependent protein kinase and lipolysis in rat adipocytes. I. Cell preparation, manipulation, and predictability in behavior.J. Biol. Chem. 1985; 260: 15122-15129Abstract Full Text PDF PubMed Google Scholar). Briefly, three male C57BL/6J mice (8–10 weeks old) (Taconic, Rockville, MD) were anesthetized using Forane® (Baxter, Deerfield, IL) and euthanized by cervical dislocation. Inguinal and epididymal fat pads (∼2 g) were harvested and placed in weighing boats containing PBS (pH 7.4) at room temperature. Fat pads were blotted dry, weighed, and minced thoroughly (∼2–3 mm pieces in diameter) in collagenase solution (3 ml/g of adipose). This mixture was transferred to a 30 ml narrow-mouthed polypropylene bottle (Nalgene, Rochester, NY) and incubated at 37°C with shaking at 220 rpm for 1 h. After digestion, the mixture was filtered through a 250 μm gauze mesh into a 50 ml conical polypropylene tube (Becton Dickinson, Franklin Lakes, NJ) and allowed to stand for 2–3 min. The infranatant containing the collagenase solution was carefully removed using a long needle and syringe. The floating layer of adipocytes was washed three times with 10 ml of the adenosine-replete AIS. The adipocyte solution was centrifuged for 30 s at 800 rpm, and the infranatant was removed and discarded. Typically, 25 (125,000 cells), 50 (250,000 cells), 100 (500,000 cells), 250 (1,125,000 cells), or 500 (2,250,000 cells) μl of packed cells was resuspended in 5 ml of AIS for subsequent distribution into assay tubes. All data were normalized to cell number. Cell counts were obtained by calculation after determining average cell sizes and TAG content according to Fine and Di Girolamo (6Fine J.B. Di Girolamo M. A simple method to predict cellular density in adipocyte metabolic incubations.Int. J. Obes. Relat. Metab. Disord. 1997; 21: 764-768Crossref PubMed Scopus (32) Google Scholar). Basal and stimulated lipolysis were determined by assaying glycerol released after cell incubation at 37°C for 1 h. After brief agitation of the suspension to obtain a homogeneous mixture of adipocytes, 200 μl was added to 5 ml polypropylene 12 × 75 mm incubation tubes containing 400 μl of AIS plus other lipolytic agents (PIA, ADA, ISO) and incubated for 1 h at 37°C. Two hundred microliters of the adipocyte suspension was also added to tubes containing AIS without lipolytic reagents and incubated at room temperature for 1–2 min to establish starting glycerol values. After incubation, 200 μl of infranatant was pipetted into microfuge tubes and stored at −20°C until assayed for glycerol. The glycerol assay used in these studies was the highly sensitive assay reported by Bradley and Kaslow (7Bradley D.C. Kaslow H.R. Radiometric assays for glycerol, glucose and glycogen.Anal. Biochem. 1989; 180: 11-16Crossref PubMed Scopus (108) Google Scholar) as adapted to 96-well plates by Brasaemle et al. (8Brasaemle D.L. Levin D.M. Adler-Wailes D.C. Londos C. The lipolytic stimulation of 3T3-L1 adipocytes promotes the translocation of cytosolic hormone-sensitive lipase to the surfaces of lipid storage droplets.Biochim. Biophys. Acta. 1999; 1483: 251-262Crossref Scopus (186) Google Scholar). Briefly, 1 μCi of γ-32PATP and 20 μl of glycerokinase (Roche, Indianapolis, IN) were added to 5 ml of stock assay buffer (2 mM MgCl2, 100 nM triethanolamine-HCl, 2 mg/ml BSA, and 120 μM ATP). Fifty microliters of known standards and glycerol samples in duplicate were mixed with 50 μl of the above assay buffer in each well of a 96-well plate and incubated at 37°C for 30 min. Next, 100 μl of acid mix (2 N HClO4 + 0.2 mM H3PO4) was added to the wells, and the plate was again incubated at 90°C for 30 min. After cooling to room temperature, 50 μl of 100 mM ammonium molybdate and 50 μl of 200 mM triethanolamine were added to the wells. The plate was centrifuged at 1,000 g for 20 min. One hundred microliters of the supernatant was removed, and radioactivity was determined with a liquid scintillation analyzer (Packard, Downers Grove, IL). Glycerol concentrations were determined based on the standard curve and expressed as nanomoles per 106 adipocytes. Data are reported as means ± SD. Data were analyzed using ANOVA, and P values were determined using SAS® software (SAS Institute, Inc., Cary, NC). Post hoc tests were performed using Bonferroni’s multiple comparisons when the overall treatment effects were found to be significant. Differences were deemed significant at P < 0.05. A comparison of recently published studies on the lipolysis of isolated primary murine adipose cells reveals a wide range of responsiveness to lipolytic stimulation (Table 1). Although several publications report stimulations of 30-fold or greater, the majority show much more modest stimulations of <10-fold, and some as little as 3-fold or less. It is difficult to evaluate such literature because many papers do not reveal the conditions under which basal activity was measured, and most do not provide cell concentration values. The data below will address the manipulations that may be used to enhance the magnitude of stimulation, either by reducing the basal value or by amplifying the stimulated values. The important variables to consider are 1) cell concentration, 2) agitation or shaking of the incubation mixture, and 3) composition of the incubation medium, especially management of the ambient adenosine.TABLE 1Conditions for lipolysis from selected studies in chronological orderAdditions to the Incubation MediumFirst Author / YearBasal ConditionsStimulated ConditionsCells AssayedaNumber of cells used for lipolysis if indicated in publication.Lipolytic StimulationbRatio of stimulated-to-basal lipolysis.Reaven / 1988 (21Reaven G.M. Chang H. Hoffman B.B. Additive hypoglycemic effects of drugs that modify free-fatty acid metabolism by different mechanisms in rats with streptozocin-induced diabetes.Diabetes. 1988; 37: 28-32Crossref PubMed Scopus (80) Google Scholar)None10−6 M ISO (60 min)105 cells/ml∼12-foldTozzo / 1997 (22Tozzo E. Gnudi L. Kahn B.B. Amelioration of insulin resistance in streptozotocin diabetic mice by transgenic overexpression of GLUT4 driven by an adipose-specific promoter.Endocrinology. 1997; 138: 1604-1611Crossref PubMed Scopus (81) Google Scholar)1 U/ml ADA, 10 μM PIA1 U/ml ADA, 10 μM PIA, 100 μM ISO (15 min)100 μl (20,000 / of the adipocyte protein fat cell lipolysis and cellular fatty acid Lipid Res. 1999; Full Text Full Text PDF PubMed Google μM ISO min)100 μl of to / J. Adipocyte metabolism in adipocyte fatty acid protein mice after by the fatty acid PubMed Scopus Google ADA, 100 ADA, 100 PIA, to / J. J.B. Chang L. of results in and in PubMed Scopus Google Scholar)1 U/ml U/ml ADA, 2 μM (60 × 106 / J. E. L. L. The adipose tissue of hormone-sensitive lipase in Res. PubMed Scopus Google Scholar)1 U/ml ADA, 10 μM PIA1 U/ml ADA, 10 μM PIA, 10 μM (60 min)100 μl of / C. J. D.L. C. et results in a mouse with adipocyte lipolysis, enhanced and resistance to PubMed Scopus Google Scholar)1 U/ml ADA, 100 nM PIA1 U/ml ADA, 100 nM PIA, 10 μM ISO (60 / lipolysis in transgenic animals the fatty acid protein in adipose Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar)1 U/ml ADA, 10 μM PIA1 U/ml ADA, 10 μM PIA, 100 μM ISO / The fat by increasing lipolysis and lipase Res. PubMed Scopus Google Scholar)None10−6 M / C. of hormone-sensitive lipase in white adipose of transgenic mice lipase activity not enhance in Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar)1 U/ml ADA, 100 PIA1 U/ml ADA, 100 PIA, 100 μM ISO / C. H. Tansey Londos C. A is essential for the translocation of hormone-sensitive lipase during lipolytic Cell Biol. PubMed Scopus Google Scholar)1 U/ml ADA, 100 nM PIA1 U/ml ADA, 10 μM ISO (60 / M. M. L. H. E. adipocyte lipolysis during stimulation, and dietary fat J. Metab. PubMed Scopus Google ADA, 10 μM ADA, 10 μM PIA, M ISO / Brasaemle D.L. of in the of lipolysis and lipid PubMed Scopus Google Scholar)1 U/ml ADA, 100 PIA1 U/ml ADA, 100 PIA, 5 (60 / J. C. effects on glucose glucose and fat cell lipolysis in and diabetic PubMed Scopus Google μM for lipolysis were performed at 37°C. ADA, adenosine PIA, of cells used for lipolysis if indicated in of stimulated-to-basal lipolysis. in a All for lipolysis were performed at 37°C. ADA, adenosine PIA, The importance of adenosine management is in which reveals glycerol in the or absence of PIA, ADA, or in cells under basal conditions when the tubes were incubated without shaking or with shaking at rpm of or and ADA from the cell incubation medium a significant increase in basal lipolysis compared with tubes in which ADA was removed or and ADA were of ADA from the incubation mixture not the stimulated lipolysis. Glycerol was to 3-fold higher with shaking at rpm compared with tubes that were incubated without especially the high basal of glycerol in the of ADA which adenosine to which is not by adenosine are two of adenosine to consider under our conditions. First, we adenosine to suppress lipolysis during the of cell isolation and adenosine will be present to cell to of adenylyl which are to that in the absence of adenosine receptor basal activity may be high, blunting the of stimulated-to-basal activity. data the we reported with rat adipocytes under we the the other the of PIA, the adenosine receptor agonist, or of ADA a adenosine receptor-mediated of basal activity, and stimulation with ISO under these adenosine conditions to to It is also that basal and stimulated are enhanced upon shaking during incubation of the cells. It be that with the cell preparation, may a wide range of from to by incubation conditions. In to our the of ADA to the incubation medium not basal lipolysis in adipocytes adipose tissue metabolism at importance of and fat Metab. Google Scholar, studies in insulin on lipolysis and in J. Google Scholar). may be to the low concentration of adipocytes in the cell incubation mixture cells per or to in isolated adipocytes cells per incubation incubated under basal or stimulated conditions. Basal lipolysis was determined in the or absence of (−)-N6-(2-phenyl-isopropyl)-adenosine (PIA), adenosine or stimulated lipolysis was determined in the of (ISO) with or without of adipocytes were incubated at 37°C without shaking or with shaking at rpm ± of a adipocyte isolation not were than the of the the data by < In our earlier studies on rat adipocytes, we not the importance of using cell concentrations during the measurement of lipolytic (3Honnor R.C. Dhillon G.S. Londos C. cAMP-dependent protein kinase and lipolysis in rat adipocytes. II. Definition of steady-state relationship with lipolytic and antilipolytic modulators.J. Biol. Chem. 1985; 260: 15130-15138Abstract Full Text PDF PubMed Google Scholar, 4Honnor R.C. Dhillon G.S. Londos C. cAMP-dependent protein kinase and lipolysis in rat adipocytes. I. Cell preparation, manipulation, and predictability in behavior.J. Biol. Chem. 1985; 260: 15122-15129Abstract Full Text PDF PubMed Google Scholar). The importance of cell concentration and agitation are in conditions (i.e., with or without the magnitude of stimulation of glycerol by ISO over basal conditions with increasing cell shaking the tubes at rpm stimulated lipolysis by in the and samples compared with tubes that were not during We that without shaking is a increase in cAMP after the of by a M. Honnor R.C. Londos C. of glucose in the isolated rat effects of lipolytic and antilipolytic Biol. Chem. Full Text PDF PubMed Google Scholar). This was known as which from fatty of cAMP and J. of free fatty acid in adipocytes in Lipid Res. Full Text PDF PubMed Google Scholar) a in fatty acid in adipocyte compared with shaking for greater mixing of the fatty acids with the medium BSA, which and the fatty acids. more the and activation of PKA a that not with (3Honnor R.C. Dhillon G.S. Londos C. cAMP-dependent protein kinase and lipolysis in rat adipocytes. II. Definition of steady-state relationship with lipolytic and antilipolytic modulators.J. Biol. Chem. 1985; 260: 15130-15138Abstract Full Text PDF PubMed Google Scholar, M. Honnor R.C. Londos C. of glucose in the isolated rat effects of lipolytic and antilipolytic Biol. Chem. Full Text PDF PubMed Google in basal and stimulated lipolysis using different adipocyte of adipocytes were incubated at 37°C without shaking or with shaking at rpm on the glycerol of a adipocyte isolation not were than the of the the data by < The overall treatment was also significant for basal lipolysis < Basal were with ADA plus and stimulated with ISO plus The values under the the number of cells per incubation tube × cells per μl shaking is a of the of the incubation and the of the incubation The shaking be such that the adipocytes are the medium and do not rise to the and the top of the the other shaking not be that cells are shaking may be determined by of the incubation the mixture present as a solution without a upper layer of adipocytes. the shaking be when a tube or is used for at optimal shaking the effects of high cell concentrations are not cell concentrations with higher medium concentrations and to the of lipolysis in adipocytes J. of free fatty acid in adipocytes in Lipid Res. Full Text PDF PubMed Google Scholar). increasing adipocyte concentration from × 106 to × 106 the of glucose to the of acids M. of cell density on in glucose metabolism by isolated J. Google Scholar). In the current the increase from to cells per milliliter from the optimal to the range of cell concentration. In optimal results were obtained with to cells per milliliter in the incubation This range is low to glycerol by the standard we use the assay which glycerol in the low adipose tissue metabolism at importance of and fat Metab. Google Scholar) and D.M. of and on and adipocyte lipolysis in Metab. Scopus Google Scholar) also be used to glycerol from adipocyte lipolysis. Data among adipocyte is a in results. It is that data be normalized according to cell number. to protein concentration is not because the vast majority of protein in a adipocyte suspension is the BSA added to the the protein by the cells is a fraction of the protein measurement of values may be because much of the present may be by cells that to the adipocytes J. of in rat adipose for cells with Lipid Res. Full Text PDF PubMed Google Scholar). Typically, of cell using values results in a the with using to obtain cell is not well in the current We were to this by and that to a of cell over the values obtained by cell number using measurement and lipid as the most to lipolysis data is to obtain a cell as by Fine and Di Girolamo (6Fine J.B. Di Girolamo M. A simple method to predict cellular density in adipocyte metabolic incubations.Int. J. Obes. Relat. Metab. Disord. 1997; 21: 764-768Crossref PubMed Scopus (32) Google Scholar). It be that the stimulations in this were obtained with relatively mice on a standard with adipocytes modest in μm in Basal lipolysis, is in in adipose tissue of and Res. PubMed Scopus Google Scholar). We also found stimulation to with adipocytes from mice a compared with animals a It also be that the basal as well as the magnitude of stimulation achieved under the conditions may not in conditions are to comparisons of adipocyte behavior in different mouse as by different The basal in the present studies are most low and are to of stimulated lipase for many years the if not the lipase during the of lipolytic stimulation C. H. mechanisms hormone-sensitive lipase and PubMed Scopus Google Scholar). the that adipocyte lipase may to this that be in the lipolytic M. et in adipose tissue is by adipose PubMed Scopus Google Scholar). Also, of the protein kinase lipolytic consider the of In careful of the lipolytic especially upon of various it is essential to the different to the lipolytic by different and C. Londos C. The of A in lipid metabolism and adipocyte PubMed Scopus Google Scholar, C. H. Tansey Londos C. A is essential for the translocation of hormone-sensitive lipase during lipolytic Cell Biol. PubMed Scopus Google Scholar). will a and reproducible method for measuring basal and lipolysis. This was by the National Institute of Diabetes and Digestive and Kidney of