The Hyperglycemia-induced Inflammatory Response in Adipocytes

Hyperglycemia is a major independent risk factor for diabetic macrovascular disease. The consequences of exposure of endothelial cells to hyperglycemia are well established. However, little is known about how adipocytes respond to both acute as well as chronic exposure to physiological levels of hyperglycemia. Here, we analyze adipocytes exposed to hyperglycemia both in vitro as well as in vivo. Comparing cells differentiated at 4 mm to cells differentiated at 25 mm glucose (the standard differentiation protocol) reveals severe insulin resistance in cells exposed to 25 mm glucose. A global assessment of transcriptional changes shows an up-regulation of a number of mitochondrial proteins. Exposure to hyperglycemia is associated with a significant induction of reactive oxygen species (ROS), both in vitro as well as in vivo in adipocytes isolated from streptozotocin-treated hyperglycemic mice. Furthermore, hyperglycemia for a few hours in a clamped setting will trigger the induction of a pro-inflammatory response in adipose tissue from rats that can effectively be reduced by co-infusion of N-acetylcysteine (NAC). ROS levels in 3T3-L1 adipocytes can be reduced significantly with pharmacological agents that lower the mitochondrial membrane potential, or by overexpression of uncoupling protein 1 or superoxide dismutase. In parallel with ROS, interleukin-6 secretion from adipocytes is significantly reduced. On the other hand, treatments that lead to a hyperpolarization of the mitochondrial membrane, such as overexpression of the mitochondrial dicarboxylate carrier result in increased ROS formation and decreased insulin sensitivity, even under normoglycemic conditions. Combined, these results highlight the importance ROS production in adipocytes and the associated insulin resistance and inflammatory response. Hyperglycemia is a major independent risk factor for diabetic macrovascular disease. The consequences of exposure of endothelial cells to hyperglycemia are well established. However, little is known about how adipocytes respond to both acute as well as chronic exposure to physiological levels of hyperglycemia. Here, we analyze adipocytes exposed to hyperglycemia both in vitro as well as in vivo. Comparing cells differentiated at 4 mm to cells differentiated at 25 mm glucose (the standard differentiation protocol) reveals severe insulin resistance in cells exposed to 25 mm glucose. A global assessment of transcriptional changes shows an up-regulation of a number of mitochondrial proteins. Exposure to hyperglycemia is associated with a significant induction of reactive oxygen species (ROS), both in vitro as well as in vivo in adipocytes isolated from streptozotocin-treated hyperglycemic mice. Furthermore, hyperglycemia for a few hours in a clamped setting will trigger the induction of a pro-inflammatory response in adipose tissue from rats that can effectively be reduced by co-infusion of N-acetylcysteine (NAC). ROS levels in 3T3-L1 adipocytes can be reduced significantly with pharmacological agents that lower the mitochondrial membrane potential, or by overexpression of uncoupling protein 1 or superoxide dismutase. In parallel with ROS, interleukin-6 secretion from adipocytes is significantly reduced. On the other hand, treatments that lead to a hyperpolarization of the mitochondrial membrane, such as overexpression of the mitochondrial dicarboxylate carrier result in increased ROS formation and decreased insulin sensitivity, even under normoglycemic conditions. Combined, these results highlight the importance ROS production in adipocytes and the associated insulin resistance and inflammatory response. Many genetic and environmental factors can lead to the development of insulin resistance. Once a degree of insulin resistance is established, decreased glucose tolerance arises and occasional bouts of hyperglycemia ensue. Hyperglycemia can in turn cause a further deterioration of insulin sensitivity in a number of tissues, such as the vascular endothelium, muscle, and adipocytes (1Brownlee M. Nature. 2001; 414: 813-820Crossref PubMed Scopus (7128) Google Scholar). In the vascular endothelium, hyperglycemia has been shown to activate protein kinase C isoforms, give rise to increased levels of glucose-derived advanced glycation end products, and to cause an increased glucose flux through the aldose reductase pathway. Normalization of mitochondrial reactive oxygen species by a number of different approaches prevents these phenomena (2Nishikawa T. Edelstein D. Du X.L. Yamagishi S. Matsumura T. Kaneda Y. Yorek M.A. Beebe D. Oates P.J. Hammes H.P. Giardino I. Brownlee M. Nature. 2000; 404: 787-790Crossref PubMed Scopus (3693) Google Scholar). In adipocytes, Tang and colleagues (3Tang S. Le-Tien H. Goldstein B.J. Shin P. Lai R. Fantus I.G. Diabetes. 2001; 50: 83-90Crossref PubMed Scopus (32) Google Scholar) have shown that a combination of hyperglycemia and hyperinsulinemia results in reduced insulin-stimulated glucose uptake that was in part because of reduced insulin receptor dephosphorylation. Gagnon and Sorisky (4Gagnon A. Sorisky A. Obes. Res. 1998; 6: 157-163Crossref PubMed Scopus (35) Google Scholar) have previously assessed the effects of low and high glucose levels on 3T3-L1 adipocytes and reported effects on insulin-mediated IRS-1 1The abbreviations used are: IRS-1, insulin reactive species 1; ROS, reactive oxygen species; GDI, GDP dissociation inhibitor; UCP1, uncoupling protein 1; 8-OHdG, 8-OH deoxyguanidine; DIC, dicarboxylate carrier; CCCP, carbonyl cyanide m-chlorophenylhydrazone; IL-6, interleukin-6; FCS, fetal calf serum; NAC, N-acetylcysteine. phosphorylation and associated phosphatidylinositol kinase activity. Lu and colleagues (5Lu B. Ennis D. Lai R. Bogdanovic E. Nikolov R. Salamon L. Fantus C. Le-Tien H. Fantus I.G. J. Biol. Chem. 2001; 276: 35589-35598Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) exposed primary rat adipocytes to hyperglycemic conditions and found reduced insulin sensitivity and increased reactive oxygen species (ROS) levels in vitro under those conditions. Similarly, Talior and colleagues (6Talior I. Yarkoni M. Bashan N. Eldar-Finkelman H. Am. J. Physiol. 2003; 285: E295-E302Crossref PubMed Scopus (147) Google Scholar) used in an ex vivo model of adipocytes isolated from animals with high fat diet-induced diabetes and demonstrated that in vitro, these adipocytes displayed significantly elevated ROS levels that could be normalized upon incubation of cells in low glucose. These adipocytes also displayed increased basal glucose uptake and reduced insulin-mediated glucose uptake. Here, we extend these observations to an in vivo setting and demonstrate increased oxidative damage in primary adipocytes. We use the 3T3-L1 cell line to further define the close link of nutrient excess, ROS production, insulin resistance, and concomitant activation of the inflammatory response in adipocytes. Materials—Thenoyltrifluoroacetone and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were obtained from Sigma; tetrakis(4-benzoic acid)porphyrin was from Calbiochem. 5-(and-6)-Chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) was from Molecular Probes (Eugene, OR). 8-OhdG-EIA Kit was from OXIS (Portland, OR). Dulbecco's modified Eagle's medium was purchased from Cellgro Inc. Murine tumor necrosis factor-α and IL-6 was purchased from BD Pharmingen. Lipopolysaccharide (from Escherichia coli) was purchased from Sigma. [1-3H]2-Deoxyglucose was purchased from Amersham Biosciences. Recombinant adenovirus vectors pAd5CMVK-NpA expressing uncoupling protein 1 (UCP1) or MnSOD were obtained as described in described in Ref. 2Nishikawa T. Edelstein D. Du X.L. Yamagishi S. Matsumura T. Kaneda Y. Yorek M.A. Beebe D. Oates P.J. Hammes H.P. Giardino I. Brownlee M. Nature. 2000; 404: 787-790Crossref PubMed Scopus (3693) Google Scholar. All other chemicals were purchased from Fisher. Cell Culture—3T3-L1 murine fibroblasts (a generous gift of Dr. Charles Rubin, Department of Molecular Pharmacology, Albert Einstein College of Medicine) were propagated and differentiated according to the protocol described in Ref. 7Engelman J.A. Berg A.H. Lewis R.Y. Lin A. Lisanti M.P. Scherer P.E. J. Biol. Chem. 1999; 274: 35630-35638Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar with the exception that media contained either 4 or 25 mm glucose and media changes were performed daily. In brief, the cells were propagated in “FCS” (Dulbecco's modified Eagle's medium containing 10% fetal calf serum (JRH Biosciences) and penicillin/streptomycin (100 units/ml each)) and allowed to reach confluence (Day–2). After 2 days (Day 0), the medium was changed to “DM1” (containing FCS and 160 nm insulin, 250 μm dexamethasone, and 0.5 mm 3-isobutyl-1-methylxanthine). Two days later (day 2), the medium was switched to “DM2” (FCS containing 160 nm insulin). After another 2 days, the cells were switched backed to FCS. Cells used between days 8 and 12 post-induction of differentiation were propagated in 5 mm glucose and used only up to passage 12. Up to that passage number, no significant differences were observed with respect to the various read outs reported here. Measurement of [1-3H]2-Deoxyglucose Uptake—Assay of [3H]2-deoxyglucose uptake was performed as described previously (8Pekala P. Kawakami M. Vine W. Lane M.D. Cerami A. J. Exp. Med. 1983; 157: 1360-1365Crossref PubMed Scopus (60) Google Scholar). Briefly, the cells were glucose starved for 30 min. Insulin was then added at the indicated concentrations for 5 min, followed by addition of the labeled glucose in the continued presence of the indicated amount of insulin. Glucose transport assays were carried out in a volume of 1 ml (Krebs-Ringer/HEPES buffer, pH 7.4) in a 3.5-cm dish for 25 min at 37 °C. The concentration of 2-deoxyglucose was 50 μm with 0.33 μCi of [3H]2-deoxyglucose/ml. After terminating the transport assay with three washes of ice-cold assay buffer, each plate was in 1 ml of and with 50 of A was for of by and were used for of protein a Glucose uptake the 30 min. at the indicated insulin concentrations the of 8 independent performed on with different cell Measurement of ROS were with medium and then in the with the μm in for min at 37 °C. The of was in an at an of nm and at nm by the ROS production was from an standard of were as described previously Diabetes. PubMed Scopus Google J. L. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google the in E. cells S. J. B. S. A. 1998; PubMed Scopus Google Scholar). The adenovirus was a gift of Dr. Albert Einstein College of adipocytes were by adenovirus at a of of as described in Ref. J. J. Res. 2001; Full Text Full Text PDF PubMed Google Scholar. was with medium containing 0.5 for min to the addition to 3T3-L1 adipocytes. After 4 the medium was with medium and cells were for an 2 of 8-OH in from adipose tissue was isolated as described in Ref. P. 1998; PubMed Scopus Google Scholar. was used to formation of After the was with mm containing 5 mm has been shown to The were with to to and then of was for the levels of the for production in adipocytes was by assay with from The was carried out according to the was in or were and used as rats were in and to a standard to to All rats were rat that of and fat with a physiological of were the in vivo rats were by of and were in the and in the of and 1 The to the of the and the was advanced to the of the was continued was of the These rats were of and was to insulin secretion and glucose was to and rats to glucose concentration to glucose concentration was at that the of the a of according to glucose the end of the rats were of The was and adipose tissue were in with in The were and by the and of the Albert Einstein College of of were with a of at was performed through glucose levels were with a glucose on a were days glucose levels and of from and tissue cells was performed with of and to was described in Ref. T. Lin S. A. PubMed Scopus Google Scholar. were performed at in the were used at concentrations of 2 The were in and at 50 from the were as was from cells and tissue the then according to the protocol concentrations were with by the OR). to the carrier were a gift of Dr. of to the of and dicarboxylate carrier were described previously Lewis R.Y. Lin Y. L. Scherer P.E. J. 1999; PubMed Scopus Google Scholar). IRS-1 and were from to the insulin receptor from and from and and from Cell dissociation and were a gift from Dr. and were described previously Lin Y. M. H. R. N. Scherer P.E. PubMed Scopus Google Scholar). were to were in or with and and were in or with and serum were by according to the of by and were performed as described previously P.E. Lisanti M.P. M. C. J. Cell Biol. PubMed Scopus Google Scholar). was performed by Insulin in 3T3-L1 differentiation protocol in use for in vitro differentiation of adipocytes for 25 mm glucose to be at of We to is to cells at physiological glucose levels as We to the cells in the presence of 4 mm glucose. the glucose levels a of medium of a medium Cells differentiated under these conditions significantly as by the reduced of differentiation to the as cells as by the induction of levels of a of or differentiation such as the of the insulin and the protein levels were the cells were differentiated in high glucose A by reveals an increased number of upon differentiation in 4 mm glucose with the reduced of levels were reduced in the cells differentiated at 4 mm Hyperglycemia Insulin in 3T3-L1 are differences between the adipocytes obtained under or hyperglycemic we the degree of insulin sensitivity in cells obtained with the differentiation both a degree of differentiation was obtained as by the induction of as previously A response to insulin was and the degree of phosphorylation of the insulin IRS-1, and was assessed phosphorylation of the insulin receptor and IRS-1 were in cells differentiated at 4 mm as by an of cell Similarly, phosphorylation was significantly in cells differentiated in 4 mm glucose. levels were in and was also for by for the Y. H. Berg A.H. Lisanti M.P. L. Scherer P.E. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). In an independent the degree of phosphorylation of the insulin receptor as well as levels of insulin receptor were assessed with results further these we assessed the phosphorylation of and on IRS-1 with a of phosphorylation on was significantly elevated in cells differentiated in 4 mm even in the basal with an response to insulin. significant differences were observed on respect to phosphorylation on associated with a in the of IRS-1 with phosphatidylinositol differences were observed on and phosphorylation is significantly increased at 25 mm glucose in the basal and further upon insulin on a induction upon insulin In differentiation of cells under hyperglycemic conditions results in significant differences in the phosphorylation of and with on at 4 mm and increased phosphorylation at 25 mm glucose. in Glucose to Glucose and a Glucose of cells to hyperglycemic damage is the to glucose uptake in response to hyperglycemic conditions (1Brownlee M. Nature. 2001; 414: 813-820Crossref PubMed Scopus (7128) Google Scholar). to endothelial adipocytes to a in basal glucose uptake upon differentiation as by a significantly increased uptake in the of insulin Furthermore, the reduced to the insulin-mediated activation of insulin a read out as insulin-stimulated glucose uptake is significantly reduced in cells differentiated in 25 mm glucose a global of the differences at the transcriptional between cells differentiated in normoglycemic hyperglycemic we performed a of in the The differences are in I. a number of are by the glucose protein kinase is was also in the insulin resistance by in was associated with a in protein kinase H. Bogdanovic E. L. Fantus I.G. A. Am. J. Physiol. PubMed Scopus Google Scholar). observations have been reported in the vascular (1Brownlee M. Nature. 2001; 414: 813-820Crossref PubMed Scopus (7128) Google Scholar). the other is a increased transcriptional of mitochondrial We have and these observations by of a of these We can a significant induction of and the mitochondrial other mitochondrial such as the and the dicarboxylate are levels of the protein protein of 30 are of is a of are significantly in the hyperglycemic Insulin and has been that activation of because of insulin resistance the formation of adipocytes J. T. Y. D. 2003; Full Text Full Text PDF PubMed Scopus Google transcriptional changes in 3T3-L1 Up Up Up Up protein Up Up Up Up glucose Up Up Up Up Up Up Up membrane Up necrosis factor receptor in a Hyperglycemia in of ROS in and in to an observations in endothelial cells and a number of other cell have shown that hyperglycemia can trigger increased levels of reactive oxygen little is known about in adipocytes. We differences in glucose levels can trigger differences in ROS levels Exposure to 30 mm glucose a in ROS could be with of oxidative such as the or tetrakis(4-benzoic In the mitochondrial also effectively the of ROS levels in 3T3-L1 adipocytes of superoxide effectively ROS Similarly, overexpression of the mitochondrial also ROS with cells with a These observations are cells are differentiated under hyperglycemic conditions or under conditions and then to hyperglycemic conditions of The to ROS are to tissue can also be observed in vivo. a model to the effects of hyperglycemia in a acute were with hyperglycemia at levels days to adipose tissue was an of we the levels of normalized to shown in in a significant in 8-OHdG, that ROS is a also in vivo. Exposure to hyperglycemia can result in the induction of pro-inflammatory such as hyperglycemic conditions have an on 3T3-L1 adipocytes as we IL-6 production in adipocytes obtained with the differentiation Cells differentiated at 25 mm levels of IL-6 ROS a in the of to an increased inflammatory we IL-6 secretion in 3T3-L1 adipocytes differentiated under hyperglycemic conditions. IL-6 levels were either upon exposure to a adenovirus or upon overexpression of A of the ROS levels by overexpression of results in a of IL-6 levels in the of these that increased ROS levels are at in the in the pro-inflammatory response in adipocytes to Insulin have shown that elevated glucose levels trigger insulin resistance. also that elevated glucose levels trigger increased ROS We to mitochondrial hyperpolarization even in the of elevated glucose is to insulin resistance. We have previously and the murine mitochondrial dicarboxylate and demonstrated that overexpression of is in adipose tissue and to a in and to hyperpolarization of Lewis R.Y. Lin Y. L. Scherer P.E. J. 1999; PubMed Scopus Google Scholar). Here, we have used to further in adipocytes and the effects on ROS production and insulin of to a in ROS levels in ROS levels is by of and by elevated glucose because ROS levels are increased to the at both 5 and 25 mm glucose in the presence of be observed upon with an adenovirus or with expressing or a to effects of high low glucose from effects to the mitochondrial membrane the effects observed upon with uncoupling the mitochondrial membrane and the concomitant in ROS in the presence of 5 mm glucose a in insulin sensitivity as by the reduced phosphorylation of the insulin receptor IRS-1 and is the that mitochondrial hyperpolarization to decreased sensitivity to insulin the effects are for or effects can be obtained by overexpression of other mitochondrial we used for the the carrier as well as the of these an with with respect to mitochondrial ROS production even overexpression was effectively as demonstrated by of and cells The Insulin the increased levels of ROS in cells differentiated under hyperglycemic conditions a on the 3T3-L1 adipocytes were differentiated in 25 mm glucose. differentiation cells were either with a or an adenovirus expressing or cells were for min with various concentrations of insulin and levels of and were shown in insulin-mediated activation of was of ROS that the differentiation of cells under hyperglycemic conditions results in changes at the or protein that are a of 2 Hyperglycemia a by of to these results obtained in a tissue are in vivo. previously hyperglycemic conditions lead to the induction of a of acute in adipocytes. Here, we extend the to a close of protein and and and In an independent we the up-regulation of acute in adipose tissue can be by ROS we the N-acetylcysteine the hyperglycemic and for the induction of of these hyperglycemic the induction of that ROS production is for the induction of pro-inflammatory transcriptional in adipose tissue in vivo as We that the glucose levels for 3T3-L1 adipocytes from the 25 mm glucose to severe hyperglycemic to the 4 mm the of differentiation of the adipocytes. such as the insulin as well as of the insulin are to the 3T3-L1 adipocytes differentiated under normoglycemic conditions a significant with respect to insulin-mediated as well as insulin-mediated glucose uptake. In we have on a of phosphorylation IRS-1 and demonstrated a significant in phosphorylation on and under hyperglycemic both of have been in a reduced J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google M. P. S. M. Y. H. Diabetes. 2003; PubMed Scopus Google Scholar). phosphorylation on that an for the of IRS-1 with phosphatidylinositol and S. S. I. E. PubMed Scopus Google Scholar) was significantly under normoglycemic conditions. In of the well effects of hyperglycemia on the production of reactive oxygen species in endothelial we on a of that demonstrate the close link of nutrient in the of and the concomitant in ROS in adipocytes. All of the in the (2Nishikawa T. Edelstein D. Du X.L. Yamagishi S. Matsumura T. Kaneda Y. Yorek M.A. Beebe D. Oates P.J. Hammes H.P. Giardino I. Brownlee M. Nature. 2000; 404: 787-790Crossref PubMed Scopus (3693) Google Scholar) up in adipocytes as we demonstrate for the in ROS levels in isolated adipocytes from hyperglycemic In in vivo we animals to hyperglycemic we demonstrate the induction of a pro-inflammatory in adipose tissue that the J. D. W. D. Du T. H. P. R. J. Full Text Full Text PDF PubMed Scopus Google M.A. N. Full Text Full Text PDF PubMed Scopus Google Scholar) and the acute A. C. M. P. N. A. J. Res. 2003; Full Text Full Text PDF PubMed Scopus Google a close to as well as Biol. 1998; PubMed Scopus Google H. H. 2000; PubMed Scopus Google Scholar). respect to the of adipose tissue J. 2003; PubMed Scopus Google D. M. M. J. 2003; PubMed Scopus Google H. D. J. A. H. J. 2003; PubMed Scopus Google we these from adipocytes or from the fat that these in response to We previously demonstrated induction of the acute under these and that production of was at the of the adipocytes Y. N. Scherer P.E. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). However, the of each cell is to that is a high degree of between adipocytes and adipose tissue with both cell for the inflammatory response A.H. Lin Y. Lisanti M.P. Scherer P.E. Am. J. Physiol. PubMed Scopus Google Scholar). of the of these factors adipose the hyperglycemic with that ROS M. J. M. Cell 2003; PubMed Scopus Google Scholar) the induction of acute with an of ROS in adipose tissue in vivo under those conditions as In tissue the increased ROS levels can be by uncoupling the transport or by the ROS by overexpression of (2Nishikawa T. Edelstein D. Du X.L. Yamagishi S. Matsumura T. Kaneda Y. Yorek M.A. Beebe D. Oates P.J. Hammes H.P. Giardino I. Brownlee M. Nature. 2000; 404: 787-790Crossref PubMed Scopus (3693) Google Scholar). we a between hyperglycemia and increased ROS be other effects that lead to decreased insulin We an that to ROS production at the of under normoglycemic conditions. We previously and the for the mitochondrial dicarboxylate Lewis R.Y. Lin Y. L. Scherer P.E. J. 1999; PubMed Scopus Google Scholar). In we that overexpression of in a cell line in a significant degree of hyperpolarization of the mitochondrial The for has been in However, is that increased uptake of by the of the transport the the of the other that other that on the We of and further the in 3T3-L1 adipocytes and demonstrated a significantly increased formation of ROS, even under normoglycemic conditions. at 4 mm these cells significantly reduced insulin sensitivity, mitochondrial ROS production to insulin resistance. is at high levels in adipocytes in other cell Lewis R.Y. Lin Y. L. Scherer P.E. J. 1999; PubMed Scopus Google a factor for the mitochondrial is of because overexpression of a number of to have an on ROS will have to levels of in adipocytes have both with respect to as a for ROS production as well as an part of the that to the for L. Y. H. B. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). We and have used 3T3-L1 cells for the of inflammatory read outs and demonstrated the pro-inflammatory of these cells Y. H. Berg A.H. Lisanti M.P. L. Scherer P.E. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google H. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Res. PubMed Scopus Google Scholar). We were to that upon the differentiation conditions to the basal inflammatory by IL-6 are reduced in these cells the that these adipocytes have differentiation further the importance of the differentiation conditions and the of the 3T3-L1 cell line that can be used as a model cell for a with a upon differentiation under conditions as well as a model for an upon differentiation under hyperglycemic conditions. the effects on ROS levels and are in the insulin are as by the that overexpression of or in cells differentiated at 25 mm insulin-mediated activation of at an that be by of of the will have to are to these and J. Cell Biol. 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