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

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 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. 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. glucose transporter 4 interleukin insulin receptor substrate 2 phosphoinositide 3-kinase p38 mitogen-activated protein kinase peroxisome proliferator-activated receptor tumor necrosis factor Dysregulation of adipose tissue has been shown to play a critical role in the etiologies of numerous pathologies, including obesity, insulin resistance, and atherosclerosis (1Kahn S.E. Hull R.L. Utzschneider K.M. Mechanisms linking obesity to insulin resistance and type 2 diabetes.Nature. 2006; 444: 840-846Crossref PubMed Scopus (3433) Google Scholar, 2Allende-Vigo M.Z. Pathophysiologic mechanisms linking adipose tissue and cardiometabolic risk.Endocr. Pract. 2010; 16: 692-698Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Adipose tissue is composed of not only adipocytes but also a variety of immune cells and an elaborate stromal-vascular matrix (3Sun K. Kusminski C.M. Scherer P.E. Adipose tissue remodeling and obesity.J. Clin. Invest. 2011; 121: 2094-2101Crossref PubMed Scopus (1221) Google Scholar, 4Anderson E.K. Gutierrez D.A. Hasty A.H. Adipose tissue recruitment of leukocytes.Curr. Opin. Lipidol. 2010; 21: 172-177Crossref PubMed Scopus (124) Google Scholar). 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-specific 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 (5Rodbell M. Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis.J. Biol. Chem. 1964; 239: 375-380Abstract Full Text PDF PubMed Google Scholar). 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 (6Foley J.E. Foley R. Gliemann J. Rate-limiting steps of 2-deoxyglucose uptake in rat adipocytes.Biochim. Biophys. Acta. 1980; 599: 689-698Crossref PubMed Scopus (47) Google Scholar–13Fantuzzi G. Adipose tissue, adipokines, and inflammation.J. Allergy Clin. Immunol. 2005; 115: 911-919Abstract Full Text Full Text PDF PubMed Scopus (1946) Google Scholar). 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 inflammation and insulin sensitivity. Ruan et al. showed that isolating adipocytes by collagenase digestion induced an acute and chronic release of the proinflammatory factors tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), respectively (14Ruan H. Zarnowski M.J. Cushman S.W. Lodish H.F. Standard isolation of primary adipose cells from mouse epididymal fat pads induces inflammatory mediators and down-regulates adipocyte genes.J. Biol. Chem. 2003; 278: 47585-47593Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Importantly, treatment with these cytokines has been shown to promote insulin resistance in 3T3-L1 adipocytes (15Ruan H. Hacohen N. Golub T.R. Van Parijs L. Lodish H.F. Tumor necrosis factor-alpha suppresses adipocyte-specific genes and activates expression of preadipocyte genes in 3T3-L1 adipocytes: nuclear factor-kappaB activation by TNF-alpha is obligatory.Diabetes. 2002; 51: 1319-1336Crossref PubMed Scopus (419) Google Scholar–19Xie L. Ortega M.T. Mora S. Chapes S.K. Interactive changes between macrophages and adipocytes.Clin. Vaccine Immunol. 2010; 17: 651-659Crossref PubMed Scopus (59) Google Scholar). Consistent with these findings, 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 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 γ (PPAR-γ) (14Ruan H. Zarnowski M.J. Cushman S.W. Lodish H.F. Standard isolation of primary adipose cells from mouse epididymal fat pads induces inflammatory mediators and down-regulates adipocyte genes.J. Biol. Chem. 2003; 278: 47585-47593Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Interestingly, expression of genes involved in insulin responsiveness were downregulated even in adipocytes isolated from TNF-α(−/−) knockout mice, suggesting that increased IL-6, not TNF-α, secretion is the more likely cause of isolation-induced inflammation and subsequent insulin desensitization (14Ruan H. Zarnowski M.J. Cushman S.W. Lodish H.F. Standard isolation of primary adipose cells from mouse epididymal fat pads induces inflammatory mediators and down-regulates adipocyte genes.J. Biol. Chem. 2003; 278: 47585-47593Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). 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-α or leptin content, is strongly inversely correlated with insulin-stimulated glucose transport in isolated adipocytes (20Bastard J-P. Maachi M. Van Nhieu J.T. Jardel C. Bruckert E. Grimaldi A. Robert J-J. Capeau J. Hainque B. Adipose tissue IL-6 content correlates with resistance to insulin activation of glucose uptake both in vivo and in vitro.J. Clin. Endocrinol. Metab. 2002; 87: 2084-2089Crossref PubMed Scopus (482) Google Scholar). 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 proinflammatory 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. Retired male Sprague Dawley breeders (∼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. Following anesthesia by CO2 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 CaCl2, 120 mM NaCl, 4 mM KH2PO4, 1 mM MgSO4, 10 mM NaCO3, 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 (≥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 ∼2 min (at ∼3 openings and closings of in of adipose tissue. The digestion was then to a and in orbital shaking incubators at at or 120 were of tissue were and of the digestion a of cells the was and to the of the digestion the cells were into a for and of stromal-vascular without the need for were in the with of or The cells were then to of adipocytes or stromal-vascular into the of adipocytes was a and The of isolated adipocytes of tissue was the on the this the isolated adipocytes were or as of adipose tissue digestion 3.3 and at 120 at were at the time the of the digestion The digestion was then a cell a the cell was into the from the of the to of tissue. with 2% BSA was to the cell to cell for the cell were then 1:1 with a solution of these were to cell with prior to of cell cells were adipocytes were for h at in containing 2% Fraction-V and pH at a 1:1 or ratio of milliliters of adipocytes to milliliters of of was to a of isolated adipocytes in of at cells from were from a of were a with a and an were with a and were measured Following the h isolated adipocytes were with or without insulin for 10 min at were then in and for min at The buffer the adipocyte was and the of cells was at were isolated for preparation at the time of digestion or at the of the h treatment with or without buffer mM pH 7.4, mM NaCl, 10 mM mM 10 1 and 2 with without 2 was to of isolated adipocytes at the of cells. 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Previous studies have shown that mechanical induces p38 which subsequently leads to increased IL-6 A. induces IL-6 expression in cells via signaling J. 2005; PubMed Scopus Google Scholar). that digestion at a rotational speed mechanical as measured by p38 phosphorylation and IL-6 Contrary to adipocytes isolated by g of adipose tissue at 30 p38 phosphorylation adipocytes isolated by at or 120 increasing the rotational speed therefore, mechanical digestion, the time to digestion it tissue at 30 min and min to digestion tissue at and 120 it tissue at min to digestion tissue at 120 Consistent with the observation that dissociation at 30 is associated with p38 at the time of digestion adipose tissue by at 30 and more IL-6 adipose tissue by at or 120 respectively into both the concentration of IL-6 at the time of digestion and the time to digestion the of IL-6 secretion was not different were at or 120 To determine how rotational speed IL-6 in adipocytes of the digestion isolated adipocytes were for h and of the stromal-vascular isolated by at 30 and more IL-6 adipocytes isolated by at or 120 respectively Importantly, the of IL-6 from adipocytes isolated by at 30 and and the to insulin sensitivity in adipocytes I. induces insulin resistance in adipocytes and is, and tumor necrosis in fat cells from Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, L. Ortega M.T. Mora S. Chapes S.K. Interactive changes between macrophages and adipocytes.Clin. Vaccine Immunol. 2010; 17: 651-659Crossref PubMed Scopus (59) Google Scholar). IL-6, has been shown to insulin sensitivity in adipocytes J. M. insulin resistance in adipocytes of insulin receptor PubMed Scopus Google Scholar, C. L. J-P. Maachi M. A. Capeau J. M. treatment with induces insulin resistance in and 2006; PubMed Scopus Google Scholar). insulin sensitivity is a in isolated primary adipocytes, to the secretion of in response to various rotational of the time of digestion the of at all rotational were the of not digestion secretion showed a to be in adipocytes isolated by at 120 with adipocytes isolated by at 30 or The concentration of by isolated adipocytes in these was the concentration of to insulin sensitivity in adipocytes J. M. insulin resistance in adipocytes of insulin receptor PubMed Scopus Google Scholar). of the for studies a primary adipocyte isolation procedure including the mincing technique, collagenase time to digestion rotational shaking digestion digestion the ratio of tissue to of digestion and the of tissue of these studies used a collagenase concentration that used in the studies or in Rodbell's studies that a collagenase concentration is to the of digestion and observation that a more rapid dissociation of adipocytes leads to attenuated IL-6 the time to digestion and IL-6 secretion between with 3.3 in a of tissue digestion conditions in a of studies a primary adipocyte isolation M. Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis.J. Biol. Chem. 1964; 239: 375-380Abstract Full Text PDF PubMed Google from were into mM glucose J.E. Foley R. Gliemann J. Rate-limiting steps of 2-deoxyglucose uptake in rat adipocytes.Biochim. Biophys. Acta. 1980; 599: 689-698Crossref PubMed Scopus (47) Google with a 30 containing BSA K. and protein of the glucose transport in fat Biol. Chem. Full Text PDF PubMed Google 2% 2 mM glucose C. protein kinase and in rat adipocytes. of with and Biol. Chem. Full Text PDF PubMed Google were with a tissue 2 mM glucose H. I. S. for of glucose transport and the of in isolated rat adipose cells and In and to the C. R. and L. C. A. R. with dissecting scissors ∼2 in 1 BSA G. Cushman S.W. that GLUT4 the of glucose transport in isolated rat adipose PubMed Google BSA J. Effects of on leptin from isolated rat 2002; PubMed Scopus Google mM glucose H. Zarnowski M.J. Cushman S.W. Lodish H.F. Standard isolation of primary adipose cells from mouse epididymal fat pads induces inflammatory mediators and down-regulates adipocyte genes.J. Biol. Chem. 2003; 278: 47585-47593Abstract Full Text Full Text PDF PubMed Scopus (125) Google M. S. heterogeneity in adipose tissue of insulin receptor is associated with in of gene Biol. Chem. Full Text Full Text PDF PubMed Scopus Google S.W. of the release of by adipose tissue, adipose tissue and adipocytes from and adipose of PubMed Scopus Google was with scissors into BSA C. C. H. between adipocyte and adipokine expression and Clin. Endocrinol. Metab. PubMed Scopus Google BSA S.K. of isolated adipocytes and isolated adipose Biol. PubMed Scopus Google the tissue into pieces, approximately or BSA et al. with surgical scissors for ∼2 min (at ∼3 openings and closings of in of adipose digestion time for rotational speed of 120 or digestion time for rotational speed of or 2% BSA with Krebs-Ringer-Henseleit data digestion time for rotational speed of 120 digestion time for rotational speed of in a with Krebs-Ringer-Henseleit data To determine the effects of collagenase concentration on the time to digestion and IL-6 secretion of rotational from an of with at and 120 were showed that adipocytes isolated with more IL-6 at h digestion adipocytes isolated with 3.3 was by a min in the of time for digestion to and a for increased IL-6 at the time of digestion adipose tissue was with 3.3 Interestingly, while in the time to digestion and IL-6 were in with 3.3 of rotational adipocytes prepared by at 120 more IL-6 even only on in the collagenase digestion data suggest that the of the time to digestion to the collagenase concentration could be for the increased IL-6 secretion at h digestion but it is likely not the only Importantly, the of cells liberated at during digestion at 120 3.3 that of the cells were liberated min and of the cells were liberated min speed and of digestion could have a negative on the and of isolated adipocytes. the metrics of were in adipocytes isolated the various cell adipocyte and insulin-stimulated was in the of adipocytes isolated with 3.3 In addition, was in the or the of of adipocytes isolated 3.3 with at or 120 was in adipocyte adipocytes were isolated 3.3 with at or 120 insulin-stimulated phosphorylation at h digestion was in adipocytes isolated 3.3 with at 120 was insulin-stimulated phosphorylation in adipocytes isolated 3.3 with at 30 and studies of isolated primary adipocytes have as the need to determine the mechanisms of metabolic including obesity and has The primary adipocyte isolation procedure used in these various studies with to the primary steps of tissue and collagenase digestion, and of stromal-vascular However, of the including the mincing technique, collagenase time to digestion rotational shaking digestion digestion the ratio of tissue to of digestion and the of tissue to studies of these not not Previous by Ruan et al. has shown that the primary adipocyte isolation process itself induces an inflammatory response as measured by secretion of TNF-α and IL-6, which have been shown to have insulin-desensitizing effects (14Ruan H. Zarnowski M.J. Cushman S.W. Lodish H.F. Standard isolation of primary adipose cells from mouse epididymal fat pads induces inflammatory mediators and down-regulates adipocyte genes.J. Biol. Chem. 2003; 278: 47585-47593Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar–19Xie L. Ortega M.T. Mora S. Chapes S.K. Interactive changes between macrophages and adipocytes.Clin. Vaccine Immunol. 2010; 17: 651-659Crossref PubMed Scopus (59) Google Scholar, H. C.M. K. Golub T.R. Lodish H.F. gene in vivo adipose tissue as an of tumor necrosis for insulin 2002; 51: PubMed Scopus Google Scholar). prior study demonstrated that the isolation process leads to the downregulation of several genes involved in insulin Considering that insulin sensitivity is a in primary adipocyte these the need to the of liberated adipocytes to that experimental physiologically that isolation-induced inflammation in primary adipocytes, as measured by IL-6 to from the of digestion time and is associated with p38 digesting at a rotational speed or 120 resulted in p38 phosphorylation with at 30 Consistent with the observation that p38 activation induces IL-6 A. induces IL-6 expression in cells via signaling J. 2005; PubMed Scopus Google digestion at these rotational also resulted in IL-6 secretion at the time of digestion and h digestion with at 30 In addition, the in IL-6 secretion at both of these time was correlated with a time to digestion The of IL-6 secretion during the digestion process was for at and 120 it at 30 to digestion at which at 120 the of the of IL-6 at the time of digestion was 30 120 that adipocytes isolated by at and were for the of time during the the in IL-6 h digestion have been to in the of IL-6 secretion between adipocytes isolated at the various rotational speeds. 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