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Endocrinology Vol. 139, No. 3 1229-1238
Copyright © 1998 by The Endocrine Society

ARTICLES

Tumor Necrosis Factor-{alpha} Causes Insulin Receptor Substrate-2-Mediated Insulin Resistance and Inhibits Insulin-Induced Adipogenesis in Fetal Brown Adipocytes1

Angela M. Valverde2, Teresa Teruel2,3, Paloma Navarro4, Manuel Benito and Margarita Lorenzo

Departamento de Bioquimica y Biologia Molecular II, Facultad de Farmacia, Universidad Complutense, 28040-Madrid, Spain

Address all correspondence and requests for reprints to: Margarita Lorenzo, Departamento de Bioquimica y Biologia Molecular II, Facultad de Farmacia, Universidad Complutense, 28040-Madrid, Spain. E-mail: mlorenzo{at}eucmax.sim.ucm.es


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Treatment of fetal brown adipocytes with 0.6 nM tumor necrosis factor (TNF)-{alpha} for 24 h resulted in a partial impairment in the expression of fatty acid synthase, glycerol-3-phosphate dehydrogenase, and glucose transporter (GLUT)-4 messenger RNAs (mRNAs), as well as in the enhancement in the cytoplasmic lipid content in response to insulin. However, the expression of the tissue-specific gene, uncoupling protein 1, is increased by the presence of TNF-{alpha}. The antiadipogenic effect of TNF-{alpha} was accompanied by a down-regulation of CCAAT/enhancer-binding protein-{alpha} and ß mRNAs and up-regulation of CCAAT/enhancer-binding protein-{delta}, with the expression of peroxisome proliferator-activated receptor-{gamma} remaining essentially unmodified. Moreover, TNF-{alpha} caused an insulin resistance on the insulin-induced glucose uptake in brown adipocytes. Pretreatment with TNF-{alpha} resulted in hypophosphorylation of the insulin receptor in response to insulin, without affecting the number of insulin receptors per cell or its molecular mass. However, insulin receptor substrate (IRS)-1 and IRS-2 signaling in response to insulin showed functional differences. Thus, TNF-{alpha} pretreatment induced a hypophosphorylation of IRS-2 but not of IRS-1. This effect leads to an impairment in the IRS-2-associated phosphatidylinositol (PI) 3-kinase activation due to a decreased association of {alpha}-p85 regulatory subunit of PI 3-kinase with IRS-2 but not in the IRS-1-associated PI 3-kinase activation in response to insulin. Our results indicate that TNF-{alpha} induced an IRS-2- but not IRS-1-mediated insulin resistance on glucose transport and lipid synthesis in fetal brown adipocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BROWN adipose tissue is the main tissue involved in nonshivering thermogenesis in mammalian newborns, and is responsible for heat production associated with the expression of the mitochondrial uncoupling protein-1 (UCP1) (1). Differentiation of brown adipose tissue also encompasses an adipogenic program related to lipid synthesis, and its accumulation results in a multilocular fat droplets phenotype (2). Rat brown adipocyte differentiation occurs in late fetal development, when the noradrenergic stimulus is not yet fully developed (2, 3). At this time of development, insulin seems to be the main signal involved in brown adipogenesis, through its ability to induce the genetic expression of metabolic genes, such as fatty acid synthase (FAS), glycerol-3-phosphate dehydrogenase (G3PD), malic enzyme, and the glucose transporter GLUT4, as well as increasing glucose uptake and lipid content (4). Moreover, the coexistence of insulin and insulin-like growth factor I (IGF-I) receptors in fetal brown adipocytes also allows IGF-I to induce adipogenic differentiation at physiological doses (4, 5). Furthermore, both insulin and IGF-I induced UCP1 gene expression in fetal brown adipocytes (6, 7). Insulin/IGF-I effects in fetal brown adipocytes are produced by the activation of their cell surface receptors, which possess tyrosine kinase activity, and upon ligand binding lead to receptor autophosphorylation (5). Insulin/IGF-I also stimulated insulin-receptor substrate-1 (IRS-1) tyrosine phosphorylation and subsequently activated phosphatidylinositol (PI) 3-kinase and glucose uptake (5, 8). However, inhibition of IGF-I-induced PI 3-kinase activity by wortmannin or LY294002 resulted in a down-regulation of differentiation genes as well as in an inhibition in the glucose uptake, indicating that PI 3-kinase is a requirement for IGF-I-induced differentiation but not for mitogenesis in fetal brown adipocytes (5).

Tumor necrosis factor (TNF)-{alpha} is a potent cytokine with pleiotropic biological effects that elicits lipolytic and antilipogenic effects in adipose tissue and produces the dedifferentiation of 3T3-L1 adipocytes (9). Long-term treatment of 3T3-F442A adipocytes with TNF-{alpha} led to down-regulation of GLUT4 and adipsin messenger RNAs (mRNAs) (10, 11, 12). TNF-{alpha} also prevents the expression of G3PD and fatty acid-binding protein aP2 genes, completely abolishing differentiation and reverting fully differentiated fat cells into fibroblasts (11, 13, 14). These effects of TNF-{alpha} are produced by preventing the expression of the peroxisome proliferator-activated receptor (PPAR){gamma}) and the CCAAT/enhancer-binding protein (C/EBP)-{alpha} transcription factors (11, 13, 15) that seem to be necessary for the induction of fully differentiated adipocytic phenotype (as reviewed in Ref.16). Moreover, TNF-{alpha} induces insulin resistance, a smaller than normal response to a given amount of insulin, in cultured cells or in whole animals (17). Thus, TNF-{alpha} produced opposing physiological effects to insulin on 3T3-L1 cells, inhibiting insulin-stimulated glucose uptake and insulin-induced adipocyte differentiation (9, 18, 19). So far, the effects of TNF-{alpha} on insulin-induced differentiation of fetal brown adipocytes remains unexplored.

Regarding insulin signaling, chronic exposure of 3T3-F442A adipocytes to TNF-{alpha} strongly impaired insulin-stimulated glucose uptake by inhibiting insulin receptor autophosphorylation and IRS-1 phosphorylation (17). In this cell type, TNF-{alpha} was shown to induce serine phosphorylation of IRS-1 and convert IRS-1 into an inhibitor of the insulin receptor tyrosine kinase activity in vitro (20). A rapid inhibition of insulin receptor autophosphorylation by TNF-{alpha} has been described in NIH 3T3 cells overexpressing the human insulin receptor, as well as in hepatoma cells (21, 22). Whether or not TNF-{alpha} could cause insulin resistance in brown adipocytes and its potential underlying molecular mechanism remains to be established. In this work, we investigated the effects of pretreatment for 24 h with TNF-{alpha} on insulin-induced adipogenic and thermogenic gene expression, on the expression of transcription factors such as C/EBPs and PPAR{gamma}, and on insulin-induced glucose uptake and lipid accumulation. Furthermore, we explored whether or not TNF-{alpha} affects the early events in insulin signaling by examining insulin receptor autophosphorylation, IRS-1 and IRS-2 phosphorylation, and its association with PI 3-kinase. Our results show that TNF-{alpha} causes insulin resistance by decreasing IRS-2 phosphorylation and IRS-2-associated PI 3-kinase activity, with IRS-1 signaling being unaffected.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Insulin and BSA (fraction V, essentially fatty acid free) were from Sigma Chemical Co. (St. Louis, MO). FCS, PBS, and culture media were from Imperial Labs. (Hampshire, UK). TNF-{alpha} and collagenase were from Boehringer Mannheim (Mannheim, Germany). RNazol B was from Biotecx Lab. (Dallas, TX). Nylon membranes were GeneScreen (NEN Research Products, Boston, MA). Autoradiographic films were Kodak X-O-MAT/AR (Eastman Kodak Co., Rochester, NY). 2-Deoxy-D[1-3H]-glucose (11.0 Ci/mmol), [125I]insulin (80 µCi/µg), [{alpha}-32P]deoxycytidine triphosphate (3000 Ci/mmol), [{gamma}32P]-ATP (3000 Ci/mmol), and the multiprimer DNA-labeling system kit were purchased from Amersham (Aylesbury, UK). All other reagents used were of the purest grade available. The complementary DNAs (cDNAs) used as probes were FAS (23), G3PD (24), GLUT4 (25), UCP1 (26), ß-actin (27), C/EBPs (28), and PPAR{gamma} (29). Antiinsulin receptor monoclonal antibody-3 (Ab-3) was purchased from Oncogene Science (Uniondale, NY). The Py20 anti-Tyr(P) monoclonal antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Antimouse IgG-agarose was from Sigma Chemical Co. Protein A-agarose was from Boehringer Mannheim. The Py72 anti-Tyr(P) mouse monoclonal antibody was the generous gift of Drs. E. Rozengurt and J. Sinnet-Smith (Imperial Cancer Research Foundation, London). For IRS-1 and IRS-2 immunoprecipitations, glutathione S-transferase (GST)-fusion protein rabbit polyclonal antibodies were the generous gift of Dr. M. White (Joslin Diabetes Center, Boston, MA). The anti-{alpha}-p85 mouse monoclonal antibody was purchased from Upstate Biotechnology (Lake Placid, NY). Wheat germ lectin coupled to Sepharose (WGA) was from LKB-Pharmacia (Uppsala, Sweden).

Cell culture
Brown adipocytes were obtained from interscapular brown adipose tissue of 20-day Wistar rat fetuses and isolated by collagenase dispersion as described (6). Cells were plated at 1.5 x 106 cells/60-mm tissue culture dishes in 2.5 ml MEM with Earle’s salts supplemented with 10% FCS. After 4–6 h of culture at 37 C, cells were rinsed twice with PBS, with 70% of the initial cells attached to the dish forming a monolayer. Cells were maintained for 20 h in a serum-free medium supplemented with 0.2% (wt/vol) BSA as described (6) and were further cultured for 24 h in the absence or in the presence of 0.6 nM TNF-{alpha} with or without insulin (1, 10, and 100 nM, as indicated in each case). In other experiments, 20-h, serum-deprived cells were pretreated with TNF-{alpha} for 24 h and then stimulated with insulin for 5 min to 1 h, as indicated in each case.

Flow cytometric analysis of Nile Red fluorescence
Cytoplasmic lipid content was determined by Nile Red fluorescence emission 530 (BP 530/30 nm) in a FACScan flow cytometer (Becton-Dickinson, San Jose, CA). Cells were detached from dishes by addition of 0.05% trypsin-0.02% EDTA, and lipid content was determined in aliquots of 2 x 105 cells after the addition of Nile Red (0.1 µg/ml) (30). Results represent mean intensities of fluorescence (obtained from the histograms of number of cells vs. intensity of fluorescence) and are expressed in arbitrary units.

RNA extraction and analysis
At the end of the culture time, cells were washed twice with ice-cold PBS and lysed directly with RNazol B, following the protocol supplied by the manufacturer for total RNA isolation (31). Total cellular RNA (10 µg) was submitted to Northern blot analysis, i.e. electrophoresed on 0.9% agarose gels containing 0.66 M formaldehyde, transferred to GeneScreen membranes using a VacuGene blotting apparatus (LKB-Pharmacia), and cross-linked to the membranes by UV light. Hybridization was in 0.25 mM NaHP04, pH 7.2, 0.25 M NaCl, 100 µg/ml denatured salmon sperm DNA, 7% SDS, and 50% deionized formamide, containing denatured 32P-labeled cDNA (106 cpm/ml) for 40 h at 42 C, as previously described (32). cDNA labeling was carried out with [{alpha}32P]deoxycytidine triphosphate to a specific activity of 109 cpm/µg DNA by using multiprimer DNA-labeling system kit. For serial hybridization with different probes, the blots were stripped and rehybridized subsequently as needed in each case. Membranes were subjected to autoradiography, and relative densities of the hybridization signals were determined by densitometric scanning of the autoradiograms in a laser densitometer (Molecular Dynamics, Sunnyvale, CA). Each Northern blot analysis was performed in duplicate samples from three independent experiments.

Measurement of the 2-deoxyglucose transport
Cells were serum-deprived for 20 h and cultured for a further 24 h in the absence or presence of TNF-{alpha}. After culture, cells were washed three times with ice-cold Krebs-Ringer-phosphate buffer (KRP) (135 mM NaCl, 5.4 mM KCl, 1.4 mM CaCl2, 1.4 mM MgSO4, and 10 mM sodium pyrophosphate, pH 7.4) and then incubated with 1 ml KRP buffer with or without insulin for 10 min at 37 C. 2-Deoxy-D[1-3H]glucose (500 nCi/ml) was added to this solution, and the incubation was continued for 5 min at 37 C. The cells were then washed three times with ice-cold KRP buffer and solubilized in 1 ml 1% SDS, as previously described (33). The radioactivity of a 200-µl aliquot was determined in a scintillation counter. Glucose transport was determined in triplicate dishes from three independent experiments.

[125I]insulin binding
Cells cultured for 20 h in a serum-free medium in either the absence or presence of TNF-{alpha} were incubated for 3 h at 20 C with 0.03 nM [125I]insulin, 1 ml of binding buffer containing 25 mM HEPES-PBS, and 1 mg/ml BSA in the absence or presence of graded concentrations of unlabeled insulin. Triplicate dishes were used for each data point. At the end of the incubation, monolayers were rinsed either with ice-cold PBS-BSA or with ice-cold 0.3 M sodium acetate, pH 4.5 (containing 0.15 M NaCl), then two more times with PBS-BSA, and then dissolved in 0.1N NaOH-1% SDS-2%Na2CO3, as previously described (4, 6). Radioactivity was counted in a Packard {gamma}-counter (Downers Grove, IL). The radioactivity associated with the cells submitted to an acid wash representing internalized 125I ligand was negligible. Total binding in the absence of competing ligand was approximately 5% of the radioactivity added in all the cell types studied. Nonspecific binding was defined as the radioactivity that remained bound in the presence of 1000 nM of unlabeled ligand, and represented approximately 10% of the total binding. Bound vs. free plots, molecular masses, and number of binding sites per cell (calculated from the Scatchard plots) were derived from three separated experiments.

Immunoprecipitations
At the end of the culture time, cells were lysed at 4 C in 1 ml of a solution containing 10 mM Tris/HCl, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 100 µM Na3VO4, 1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride, pH 7.6 (lysis buffer). Lysates were clarified by centrifugation at 15,000 x g for 10 min, and the supernatants were transferred to a fresh tube. After protein content determination, equal amounts of protein (600 µg) were immunoprecipitated at 4 C with monoclonal antibodies anti-Tyr(P) (Py72) and antiinsulin receptor (Ab-3), or with polyclonal antibodies against {alpha}p85 regulatory subunit of PI 3-kinase, IRS-1 and IRS-2. The immune complexes were collected on antimouse IgG-agarose beads or on protein A-agarose beads. Immunoprecipitates were washed three times with lysis buffer and extracted for 10 min at 95 C in 2 times SDS-PAGE sample buffer (200 mM Tris/HCl, 6% SDS, 2 mM EDTA, 4% 2-mercaptoethanol, 10% glycerol, pH 6.8), and analyzed by SDS-PAGE as described in the figure legends.

Western blotting
After SDS-PAGE, proteins were transferred to Immobilon membranes (Millipore Corp., Bedford, MA), were blocked using 5% nonfat dried milk in TBS (10 mM Tris/HCl, 150 mM NaCl, pH 7.5), and incubated overnight with several antibodies as indicated in 0.05% Tween-20, 1% nonfat dried milk in 10 ml TBS. Immunoreactive bands were visualized using the enhanced chemiluminiscence (ECL) Western blotting protocol (Amersham).

In vitro kinase assay
The anti-Tyr(P) immune complexes were incubated in 20 µl buffer containing 20 mM HEPES, 3 mM MnCl2, 10 mM MgCl2, and 20 µCi [{gamma}32P]ATP (in a final concentration of 5 µM) for 15 min at room temperature, as previously described (34). The complexes were washed twice with ice-cold PBS and then resuspended in 2x SDS-PAGE sample buffer and analyzed by SDS-PAGE. The separated proteins were dried in the gel and the incorporation [32P]phosphate into protein was visualized by autoradiography and quantitated by scanning laser densitometry.

Purification and phosphorylation of the insulin receptor
Serum-deprived cells pretreated for 24 h with 0.6 nM TNF-{alpha} were solubilized in lysis buffer as described above. Solubilized receptors were added to 1 ml WGA-Sepharose and rotated end over end for 2 h at room temperature. After extensive washing with a buffer containing 50 mM Tris (pH 7.4), 0.05% Triton X-100, 100 mM NaCl, 2.5 mM KCl, and 1 mM CaCl2, receptors were eluted with 300 µl of the same buffer supplemented with 0.3 M N-acetylglucosamine. Portions containing 10 µg WGA-purified receptors were preincubated for 1 h at room temperature with 10 nM insulin. The phosphorylation reaction was performed as indicated. After 15 min on ice, phosphorylation was stopped, and receptors were immunoprecipitated with the Py72 anti-Tyr(P) antibody and separated by SDS-PAGE. The separated proteins were dried in the gel and the incorporation of [32P]phosphate into protein was visualized by autoradiography and quantitated by scanning laser densitometry.

PI 3-kinase activity
PI 3-kinase activity was measured by in vitro phosphorylation of phosphatidylinositol as previously described (35). At the end of the culture time, cells were washed with ice-cold PBS, solubilized in lysis buffer containing leupeptin (10 µg/ml), aprotinin (10 µg/ml) and 1 mM phenylmethylsulfonyl fluoride. Lysates were clarified by centrifugation at 15,000 x g for 10 min at 4 C, and proteins were immunoprecipitated with the anti-IRS-1 or the anti-IRS-2 antibodies. The immunoprecipitates were washed successively in PBS containing 1% Triton X-100 and 100 µM Na3VO4 (twice), 100 mM Tris (pH 7.5) containing 0.5 M LiCl, 1 mM EDTA and 100 µM Na3VO4 (two times), and 25 mM Tris (pH 7.5) containing 100 mM NaCl and 1 mM EDTA (twice). To each pellet 25 µl 1 mg/ml L-{alpha}-phosphatidylinositol/L-{alpha}-phosphatidyl-L-serine sonicated in 25 mM HEPES (pH 7.5) and 1 mM EDTA were added.

The PI 3-kinase reaction was started by the addition of 100 nM [{gamma}32P]-ATP (10 µCi) and 300 µM ATP in 25 µl of 25 mM HEPES, pH 7.4, 10 mM MgCl2, and 0.5 mM EGTA. After 15 min at room temperature, the reaction was stopped by the addition of 500 µl CHCl3-methanol (1:2) in a 1% concentration of HCl plus 125 µl chloroform and 125 µl HCl (10 mM). The samples were centrifuged, and the lower organic phase was removed and washed once with 480 µl methanol-100 mM HCl plus 2 mM EDTA (1:1). The organic phase was extracted, dried in vacuo, and resuspended in chloroform. Samples were applied to a a silica gel TLC plate (Whatman, Clifton, NJ). TLC plates were developed in propanol-1-acetic acid (2N; 65:35 vol/vol), dried, visualized by autoradiography, and quantitated by scanning laser densitometry.

Protein determination
Protein determination was performed by the Bradford dye method (36) using the Bio-Rad reagent (Bio-Rad, Richmond, CA) and BSA as the standard.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TNF-{alpha} down-regulates expression of insulin-induced adipogenic genes in fetal brown adipocyte primary cultures
TNF-{alpha} produced opposing effects to insulin in 3T3-L1 cells, inhibiting insulin-induced adipogenic differentiation (9, 18, 19). We first characterized the effect of TNF-{alpha} treatment on insulin-induced fetal brown adipocyte gene expression, exploring the expression of genes encoding enzymes directly involved in lipid synthesis such as FAS and G3PD, and genes indirectly involved in adipogenesis such as the insulin-regulated GLUT4 and the tissue-specific thermogenic marker UCP1. Cells (after 4 h of attachment in FCS medium) were cultured for 20 h in a serum-free medium and further cultured for 24 h either in the absence or presence of 0.6 nM TNF-{alpha} either in combination or not with different doses of insulin (1 and 10 nM). The expression of the adipogenic and thermogenic genes was studied by Northern blot, as depicted in a representative experiment that is shown in Fig. 1Go; the resulting densitometric analysis is shown in Table 1Go. Treatment with TNF-{alpha} by itself decreased the basal expression of FAS and GLUT4 mRNAs but had no significant effect on the basal expression of G3PD. Insulin treatment produced a dose-dependent induction of adipogenic genes, as well as a dramatic accumulation of GLUT4 mRNA, in agreement with the data previously described (4). However, the treatment with TNF-{alpha} completely abolished the 1 nM insulin effect on the expression of adipogenic genes and decreased FAS mRNA levels by 75% and G3PD mRNA levels by 55% at 10 nM insulin. Moreover, TNF-{alpha} caused down-regulation on the expression of GLUT4 induced by insulin (Fig. 1AGo and Table 1Go). However, ß-actin mRNA content remained essentially unmodified throughout the different treatments (Fig. 1AGo). Regarding UCP1 mRNA expression, TNF-{alpha} up-regulated UCP1 gene expression by itself, although to a lower extent than insulin 10 nM, whereas the combined presence of insulin and TNF-{alpha} produced an additive effect on UCP1 mRNA accumulation (Fig. 1BGo and Table 1Go).



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  		Figure 1. TNF-{alpha} effects in insulin-induced adipogenic and thermogenic gene expression in fetal brown adipocyte primary cultures. Fetal brown adipocytes (20 h serum deprived) were cultured for 24 h in a serum-free medium in both absence (C) and presence of 0.6 nM TNF-{alpha} (T) combined with or without insulin (i) (1 and 10 nM). Total RNA (10 µg) was submitted to Northern blot analysis and hybridized with labeled FAS, G3PD, GLUT4, and ß-actin cDNAs (A) or with UCP1 cDNA (B). A final hybridization with 18S ribosomal RNA (rRNA) cDNA was performed for normalization. Autoradiograms from a representative experiment out of three are shown.

 

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  		Table 1. Densitometric analysis of mRNA expression in fetal brown adipocytes

 
TNF-{alpha} inhibits insulin-induced cytoplasmic lipid content in fetal brown adipocytes
Because TNF-{alpha} down-regulated the expression of adipogenic genes induced by insulin, we studied Nile Red fluorescence [a sensitive detector of cytoplasmic lipid content (37)], to assess the role of TNF-{alpha} in the overall adipogenic program. Brown adipocytes were cultured as described in Fig. 1Go, and at the end of the culture period Nile Red fluorescence was analyzed in the flow cytometer and quantitated in arbitrary units, as shown in a representative experiment in Table 2Go. Treatment for 24 h with 0.6 nM TNF-{alpha} by itself significantly decreased the basal lipid content by a 25%. The presence of 10 nM insulin in the culture medium for 24 h increased by 3-fold the lipid content detected in control cells. However, when cells were treated with 0.6 nM TNF-{alpha} together with 10 nM insulin, their lipid content decreased by 50%, in agreement with the partial inhibitory effect observed above in the accumulation of adipogenic mRNAs under the same experimental conditions.


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  		Table 2. TNF-{alpha} inhibits insulin increase in cytoplasmic lipid content in fetal brown adipocytes

 
TNF-{alpha} down-regulates C/EBP{alpha} and -ß and up-regulates C/EBP{delta} in fetal brown adipocytes; PPAR{gamma} expression remains essentially unmodified
Transcription factors play an important role in the differentiation processes, more specifically the expression of the PPAR{gamma} and the C/EBPs seems to be necessary in the induction of fully differentiated adipocyte phenotype (16). Furthermore, both families of transcription factors seem also to be involved in thermogenic differentiation, because C/EBP{alpha} and -ß and PPAR{gamma} have been proposed as transcriptional activators of the UCP1 gene promoter (38, 39). Thus, we studied the effect of TNF-{alpha} treatment of brown adipocytes on C/EBP mRNA accumulation by Northern blot as shown in a representative experiment in Fig. 2AGo; the resulting densitometric analysis is shown in Table 1Go in cells cultured under the same experimental conditions seen above. Brown adipocytes cultured under basal conditions expressed the three isoforms of C/EBP transcription factors, i.e. {alpha}, ß, and {delta}. TNF-{alpha} per se decreased basal expression of C/EBP{alpha} and ß, and indeed increased C/EBP{delta} mRNA accumulation. Insulin treatment induced C/EBP{alpha} and ß mRNA content at 10 nM, with this effect being totally precluded by the treatment with TNF-{alpha}. The expression of C/EBP{delta} remained essentially unmodified under insulin treatment, increasing, however, in the combined presence of 1 nM insulin and TNF-{alpha} (Fig. 2AGo and Table 1Go).



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  		Figure 2. TNF-{alpha} effects on expression of C/EBP and PPAR{gamma} mRNAs. Fetal brown adipocytes (20 h serum deprived) were cultured for 24 h in a serum-free medium in both absence and presence of insulin (1 and 10 nM) either with or without 0.6 nM TNF-{alpha}. Total RNA (10 µg) was submitted to Northern blot analysis and hybridized with labeled C/EBP {alpha}, ß, and {delta} (A) or PPAR{gamma} cDNAs (B). A final hybridization with 18S ribosomal RNA cDNA was performed for normalization. Autoradiograms from a representative experiment out of three are shown.

 
The expression of PPAR{gamma} was studied in cells cultured for 24 h in the absence or presence of 0.6 nM TNF-{alpha} and insulin (10 nM), as shown in a representative experiment (Fig. 2BGo) and in Table 1Go. Brown adipocytes cultured under basal conditions expressed PPAR{gamma} mRNA. Nevertheless, no significantly changes on PPAR{gamma} mRNA expression were observed under treatment with either TNF-{alpha} or insulin or both combined compared with control cells (Fig. 2BGo).

TNF-{alpha} inhibits insulin-stimulated glucose uptake in fetal brown adipocytes
TNF-{alpha} inhibits insulin-stimulated glucose uptake in 3T3-L1 cells (18, 19). The fact that glucose transport is induced in fetal brown adipocytes on insulin stimulation (4), prompted us to investigate whether this effect could be blocked by TNF-{alpha} in those cells. Cells were serum deprived for 20 h and further cultured for 24 h in the presence or absence of 0.6 nM TNF-{alpha} before stimulation for 15 min with insulin at the doses indicated (Table 3Go). Glucose uptake was measured during the last 5 min of culture as described in Materials and Methods. Results from three independent experiments are expressed as disintegrations per minute per microgram of protein. Insulin stimulated basal glucose-uptake by 3-fold, with the effect being observed at all the doses tested. Glucose uptake was significantly higher (34%) in cells pretreated with TNF-{alpha} for 24 h compared with untreated cells. However, 1 nM insulin did not significantly induced glucose uptake under pretreatment with TNF-{alpha}, and only at higher doses (10 and 100 nM) produced a 1.5-fold increase in the glucose uptake, an effect 50% lower than that observed in TNF-{alpha} untreated cells (Table 3Go).


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  		Table 3. Effect of TNF-{alpha} on insulin-stimulation of glucose uptake in fetal brown adipocytes

 
TNF-{alpha} inhibits insulin-induced insulin-receptor ß-subunit tyrosine autophosphorylation
To define the underlying molecular mechanism of the insulin resistance observed on treatment with TNF-{alpha} in fetal brown adipocytes, we decided to investigate the early events in the insulin signaling pathway. Serum-starved brown adipocytes were pretreated for 24 h with or without 0.6 nM TNF-{alpha} and then incubated in the absence or presence of insulin for 1 h or 5 min, and insulin-receptor ß-chain autophosphorylation analyzed by several methods (Fig. 3Go). First, we determined the level of the tyrosine phosphorylation in purified insulin receptors. WGA-purified proteins from cells pretreated with or without TNF-{alpha} were stimulated with or without 10 nM insulin for 1 h, and then the in vitro kinase assay was performed as described in Materials and Methods, and the insulin receptors were immunoprecipitated with the Py72 anti-Tyr(P) antibody (Fig. 3AGo). Insulin-treated brown adipocytes showed a marked increase in the insulin-receptor ß-subunit tyrosine autophosphorylation as compared with nontreated cells. This insulin receptor ß-chain autophosphorylation was greatly impaired by TNF-{alpha} pretreatment. No insulin receptor tyrosine phosphorylation band was observed in control cells.



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  		Figure 3. TNF-{alpha} inhibits insulin-induced insulin-receptor ß-chain tyrosine autophosphorylation. Serum-deprived fetal rat brown adipocytes were preincubated for 24 h in absence or presence of 0.6 nM TNF-{alpha} followed by treatment for 5 min with 10 nM insulin (ins or ins + TNF-{alpha}) except in A where insulin treatment was for 1 h. Control cells (C or CTNF-{alpha}) were not treated with insulin. A, Equal amounts of WGA-purified proteins (10 µg) were submitted to an in vitro kinase assay as described in Materials and Methods, and insulin receptors were immunoprecipitated with Py72 anti-Tyr(P) antibody. B, Cells were lysed and immunoprecipitates prepared using Py72 monoclonal anti-Tyr(P) antibody and assayed for in vitro kinase activity. Proteins phosphorylated in immune complexes from both experiments shown in A and B were separated by SDS-PAGE and gels were dried and subjected to autoradiography. C, Cells were lysed and immunoprecipitates prepared using monoclonal antiinsulin receptor antibody. Immune complexes were separated by SDS-PAGE followed by transfer of proteins to Immobilon membranes (Millipore) and Western blotting with Py20 anti-Tyr(P) antibody. Specific proteins were detected by ECL. Position of ß-chain of insulin receptor is indicated by an arrowhead in A, B, and C. Positions of molecular weight markers (x 10-3) are shown on left of each panel. Results shown are representative of at least three independent experiments. Other experimental details are described in Materials and Methods.

 
In another set of experiments, we pretreated brown adipocytes for 24 h with TNF-{alpha}, and then cells were stimulated either in the absence of presence of 10 nM insulin for 5 min, after which whole-cell lysates (600 µg protein) were subjected to immunoprecipitation with the Py72 anti-Tyr(P) antibody. The immunoprecipitates were submitted to an in vitro kinase assay, as described in Materials and Methods (Fig. 3BGo). The presence of 10 nM insulin in the culture medium caused a marked increase in the insulin receptor 32 ß-subunit tyrosine phosphorylation as compared with nontreated cells. In the cells pretreated with TNF-{alpha}, the insulin receptor ß-subunit was hypophosphorylated on insulin stimulation (Fig. 3BGo).

Alternatively, the autophosphorylation of the insulin receptor was studied by direct immunoprecipitation with the antiinsulin receptor ß-subunit antibody (Fig. 3CGo). Thus, primary fetal brown adipocytes pretreated for 24 h with TNF-{alpha} were stimulated with/without 10 nM insulin for 5 min at 37 C, extracted as described in Materials and Methods, immunoprecipitated with the antiinsulin receptor monoclonal antibody, and then the resulting immune complexes were analyzed by Western blotting with the Py20 anti-Tyr(P) antibody. As shown in Fig. 3CGo, the insulin-receptor tyrosine phosphorylation induced by insulin was greatly impaired in TNF-{alpha} pretreated cells. These results were consistent with those found in the in vitro kinase assays seen above.

As an additional step, we determined whether the alterations in the insulin receptor autophosphorylation described above could be due to a reduced number of insulin receptors after pretreatment for 24 h with 0.6 nM TNF-{alpha}. However, quantitation of the insulin receptors by radioligand binding using [125I]insulin revealed an equivalent number of specific insulin binding sites per cell (36,000 ± 8,000) with a similar affinity (kDa = 18.8 ± 4.7 nM) on the surface of primary brown adipocytes under pretreatment with TNF-{alpha} than in primary brown adipocytes under basal culture conditions (35,000 ± 9,500 binding sites per cell; kDa = 16.9 ± 6.3 nM).

TNF-{alpha} inhibits insulin-induced IRS-2 phosphorylation in fetal brown adipocytes
The next step was to compare the effect of acute insulin treatment on inducing tyrosine phosphorylation of IRS-1 and IRS-2 in fetal brown adipocytes. After 20 h of serum starvation, cells pretreated with or without TNF-{alpha} were incubated in the absence or presence of insulin for 5 min at 37 C. Cell lysates (600 µg protein) were then immunoprecipitated with anti-IRS-1 or anti-IRS-2 polyclonal antibodies as described in Materials and Methods, and the immune complexes were subjected to Western blotting analysis with Py20 anti-Tyr(P) antibody. As shown in Fig. 4AGo, no tyrosine phosphorylation of either IRS-1 or IRS-2 was found in cells pretreated with or without TNF-{alpha} in the absence of insulin. However, there was a huge increase (>20-fold) in the tyrosine phosphorylation of the 180-kDa band corresponding to IRS-1 on insulin stimulation (Fig. 4AGo, left panel). Moreover, a marked increase (16-fold) in the tyrosine phosphorylation of the 190-kDa band corresponding to IRS-2 was observed on insulin treatment (Fig. 4AGo, right panel). In cells pretreated with TNF-{alpha}, IRS-1 tyrosine phosphorylation was increased by 2-fold on insulin stimulation compared with the phosphorylation observed under insulin stimulation alone. However, IRS-2 tyrosine phosphorylation was strongly impaired (>60% of inhibition) in cells pretreated with TNF-{alpha} on insulin stimulation compared with insulin stimulation alone. A possible explanation for the altered IRS phosphorylation in the presence of TNF-{alpha} would be changes at the level of IRS-1/IRS-2 expression. To assess this, equals amount of protein from cells either untreated or 24 h pretreated with TNF-{alpha} were submitted to direct Western blot analysis with the anti-IRS-1/IRS-2 antibodies. As depicted in Fig. 4BGo, both IRS-1 and IRS-2 protein levels are similar in control cells. However, pretreatment for 24 h with TNF-{alpha} doubled the amount of IRS-1, without affecting the expression of IRS-2 compared with untreated cells. The observed increased IRS-1 tyrosine phosphorylation in cells pretreated with TNF-{alpha} on acute insulin stimulation could be due to the higher IRS-1 protein content found under this experimental condition.



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  		Figure 4. TNF-{alpha} inhibits insulin-induced IRS-2 tyrosine phosphorylation. Serum-deprived fetal rat brown adipocytes were preincubated for 24 h in absence or presence of 0.6 nM TNF-{alpha} followed by treatment for 5 min with 10 nM insulin. A, Cells were then lysed and immunoprecipitates (600 µg protein) were prepared using GST-fusion protein polyclonal antibodies against IRS-1 and IRS-2. Immune complexes were analyzed by SDS-PAGE followed by transfer of proteins to Immobilon and Western blotting with Py20 anti-Tyr(P) antibody. Specific proteins were detected by ECL. Position of IRS-1 and IRS-2 are indicated by an arrowhead. Positions of molecular weight markers (x 10-3) are shown on left. B, Equal amount of proteins (30 µg) from brown adipocytes preincubated for 24 h either in absence or presence of TNF-{alpha} were submitted to SDS-PAGE followed by Western blot analysis with anti-IRS-1/IRS-2 antibodies. Specific proteins were detected by ECL, and positions of IRS-1 and IRS-2 are indicated by an arrowhead. All results shown are representative of at least three independent experiments.

 
TNF-{alpha} inhibits insulin-induced IRS-2-associated PI 3-kinase activity in fetal brown adipocytes
In our laboratory, we recently established the importance of PI 3-kinase activity in IGF-I-induced differentiation of brown adipocytes (5). In this study, we further investigated whether PI 3-kinase activity associated to IRS-1 or IRS-2 could be altered in cells pretreated with TNF-{alpha} on insulin stimulation. A representative TLC experiment is shown in Fig. 5AGo (upper panel), meanwhile means ± SEM of PI 3-kinase activities from four independent experiments are shown in Fig. 5AGo (lower panel). Cells pretreated with or without TNF-{alpha} were immunoprecipitated with anti-IRS-1 or anti-IRS-2 antibodies under identical conditions as described in Fig. 4Go. The resulting immune complexes were assayed for PI 3-kinase activity. As shown in Fig. 5AGo, primary brown adipocytes showed no detectable PI 3-kinase activity from IRS-1 immunoprecipitates under control conditions, regardless the presence or absence of TNF-{alpha}. On treatment with 10 nM insulin, PI 3-kinase activity markedly increased in IRS-1 immunoprecipitates, with this activity increasing under pretreatment with TNF-{alpha}. PI 3-kinase activity from IRS-2 immunoprecipitates under control conditions or TNF-{alpha} pretreatment was not detectable, although on acute treatment with 10 nM insulin, PI 3-kinase activity markedly increased from IRS-2 immunoprecipitates to a similar extent than from IRS-1 immunoprecipitates. In contrast, TNF-{alpha} pretreatment resulted in a marked decrease (50% of inhibition) of IRS-2-associated PI 3-kinase activity on insulin stimulation (Fig. 5AGo). Finally, we investigated whether these changes in PI 3-kinase activity associated with either IRS-1 or IRS-2 under TNF-{alpha} pretreatment were due to altered association of the {alpha}-p85 regulatory subunit of PI 3-kinase with IRSs (Fig. 5BGo). Cells treated as described above were immunoprecipitated with anti-IRS-1 or anti-IRS-2 antibodies and submitted to Western blot analysis with the anti-{alpha}-p85 monoclonal antibody. No p85 association to either IRS-1 or IRS-2 was found in control cells in the absence or presence of TNF-{alpha}. Nevertheless, under insulin stimulation, {alpha}-p85 was associated to both IRS-1 and IRS-2 to a similar extent. However, TNF-{alpha} pretreatment before the stimulation with insulin increased the association of p85 to IRS-1 and decreased the association of p85 to IRS-2 compared with insulin effects. These results on p85 association to IRSs are in agreement with the changes observed in PI 3-kinase associated activity.



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  		Figure 5. Inhibition by TNF-{alpha} of PI 3-kinase activity in anti-IRS-2 immunoprecipitates from insulin-treated brown adipocytes. A, Serum-deprived fetal rat brown adipocytes were preincubated for 24 h in absence or presence of 0.6 nM TNF-{alpha} followed by treatment for 5 min with 10 nM insulin. Cells were then lysed and immunoprecipitates (600 µg of protein) were prepared using GST-fusion protein polyclonal antibodies against IRS-1 and IRS-2. Immune complexes were washed and immediately used for an in vitro PI 3-kinase assay as described in Materials and Methods. Conversion of PI to PI phosphate in presence of [{gamma}32P]ATP was analyzed by TLC (upper panel). Corresponding autoradiograms were quantitated by scanning densitometry. Results are expressed as arbitrary units of PI 3-kinase activity and are means ± SEM from four independent experiments. Statistical analysis by Student’s paired t test between values in presence of insulin + TNF-{alpha} vs. insulin is represented by an asterisk; *, P < 0.05; **, P < 0.01 (lower panel). B, Cells were cultured and immunoprecipitated against IRS-1 and IRS-2 as described above. Immune complexes were analyzed by SDS-PAGE followed by transfer of proteins to Immobilon and Western blotting with {alpha}-p85 monoclonal antibody. Specific proteins were detected by ECL.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IGF-I/insulin induces adipogenic- and thermogenic-related gene expression in fetal brown adipocytes (4, 7). The treatment with TNF-{alpha} led to a severe insulin resistance on the expression of adipogenic genes and GLUT4 gene and also on the lipid synthesis, with this effect being partially ameliorated at higher concentrations of insulin. However, TNF-{alpha} did not impair the insulin effect on UCP1 gene expression. In fact, TNF-{alpha} produced a small accumulation in UCP1 mRNA either in the absence or presence of insulin. Our results clearly indicate that TNF-{alpha} specifically regulates gene expression in brown adipocytes, down-regulating adipogenic genes and GLUT4 gene in a similar fashion as in white fat and 3T3-L1 cells (9, 10, 11, 12, 18, 19) but up-regulating the tissue-specific thermogenic marker UCP1. Because the antiadipogenic effects of TNF-{alpha} have been related to a negative regulation of C/EBP{alpha} and PPAR{gamma} transcription factors in adipocytic differentiated fibroblasts, we previously investigated the expression of these transcription factors in brown adipocytes (11, 13, 15). The three canonical C/EBP isoforms are expressed in fetal brown adipocytes, with C/EBP{alpha} and ß being positively modulated by insulin. In this study, treatment with TNF-{alpha} down-regulated the endogenous expression of C/EBP{alpha} and ß mRNA. In addition, TNF-{alpha} impaired the insulin effect on C/EBP{alpha}-ß gene expression. These results are in agreement with those reported in TA1 and 3T3-L1 adipocytes (11, 13). However, C/EBP{delta} mRNA accumulation is regulated in a reciprocal fashion to C/EBP{alpha} and -ß by TNF-{alpha}. PPAR{gamma} gene is highly expressed in brown adipocytes, with its expression not being modulated by the presence of insulin or TNF-{alpha} in the long term in fetal brown adipocytes. Our results differ from those observed by Zhang et al. (15) in 3T3-L1 cells, who found the maximal anti-PPAR{gamma} effect of TNF-{alpha} at 24 h. Our results clearly point out that the adipogenic effect of insulin as well as the antiadipogenic effect induced by TNF-{alpha} on insulin action are mediated by the modulation of C/EBP{alpha} and ß expression but not by PPAR{gamma} expression in fetal brown adipocytes.

Besides impairing long-term insulin effects on adipogenesis, TNF-{alpha} induced an insulin resistance on the early events of insulin signaling and also on the insulin-induced glucose uptake in fetal brown adipocytes. As demonstrated by several approaches, pretreatment with TNF-{alpha} strongly inhibits insulin-induced tyrosine autophosphorylation of the ß-chain insulin receptor in brown adipocytes in a similar fashion as in adipocytic cell lines, fibroblasts, and hepatoma cells (17, 21, 22). This effect cannot be accounted for the presence of a lower number of insulin binding sites or altered insulin receptor affinity under treatment with TNF-{alpha}. Moreover, we explored the effect of TNF-{alpha} on insulin-induced phosphorylation of IRS-1 and IRS-2. In a previous work (5), we described that insulin induced tyrosine phosphorylation of IRS-1 in fetal brown adipocytes in culture. In this study, we show for the first time that in fetal brown adipocytes, both IRS-1 and IRS-2 turned out to be tyrosine phosphorylated in a similar extent on insulin stimulation, suggesting that both insulin substrates are involved in the insulin signaling pathways. Indeed, brown adipocytes constitute a different scenario as compared with primary white adipocytes and differentiated 3T3-L1 fibroblasts, in which IRS-2 tyrosine phosphorylation and IRS-2-associated PI 3-kinase activation on insulin stimulation was shown to be almost undetectable (40). More importantly, pretreatment with TNF-{alpha} blunted the IRS-2 signaling in response to insulin without affecting IRS-2 protein expression. However, IRS-1 signaling was fully active in response to insulin under pretreatment with TNF-{alpha}. Moreover, this pretreatment for 1 day with TNF-{alpha} causes up-regulation of IRS-1 expression in fetal brown adipocytes. Several potential mechanisms have been proposed to explain the inhibition of insulin signaling induced by TNF-{alpha} in different cell types. A decreased insulin-induced IRS-1 tyrosine phosphorylation has been observed in differentiated 3T3-L1 cells chronically treated (3–5 days) with TNF-{alpha}, proposing that TNF-{alpha} in murine cultured adipocytes induce serine phosphorylation of IRS-1 and convert IRS-1 into an inhibitor of the insulin receptor tyrosine kinase activity in vitro (20). Moreover, this chronic treatment causes down-regulation of IRS-1 expression (12). Acute TNF-{alpha} treatment induces serine phosphorylation of IRS-1 through inhibition of serine phosphatases in rat hepatoma cells; this increased serine phosphorylation interferes with insulin-induced tyrosine phosphorylation of IRS-1 and impairs insulin effect (41). Moreover, in this cell model, TNF-{alpha} increased abundance of SH2-phosphotyrosine phosphatases (42). All these effects of TNF-{alpha} inhibiting the insulin signaling seem to be mediated through the p55 TNF receptor by activating the sphingomyelinase. The addition of exogenous sphingomyelinase and ceramides to IR/IRS-1-reconstituted myeloid cells mimicked the inhibitory effect of TNF-{alpha} on insulin signaling, although IR/IRS-2-reconstituted 32D cells were resistant to this inhibition (43). However, a recent study involved IRS-2 and not IRS-1 in the impairment of IGF-I action in bovine fibroblasts under pretreatment with insulin and interleukin-4 (44). Our results strongly suggest functional differences between IRS-1 and IRS-2 in a cell system in which both substrates compete for the insulin receptor on insulin stimulation. Whether or not IRS-2 could be of critical importance to the understanding of insulin-resistance states produced by TNF-{alpha} remains to be established.

Tyrosine-phosphorylated insulin receptor substrates bind and activate several signaling molecules, including the 85-kDa regulatory subunit of the PI 3-kinase (45, 46, 47). The NH2-terminal SH2 domain of p85 can bind to both IRS-1 and IRS-2, and this interaction results in the activation of the PI 3-kinase (40). Recent findings indicate that PI 3-kinase is required for the movement of glucose transporter to the cell membrane in both white and brown adipose tissues and muscle cells, as well as the importance of PI 3-kinase activity for IGF-I-induced differentiation of brown adipocytes and myoblasts (5, 48, 49, 50, 51). In this study, we found that insulin induced IRS-1- and IRS-2-associated PI 3-kinase activity. Cells pretreated with TNF-{alpha} showed an impairment in IRS-2-associated PI 3-kinase activation on insulin stimulation. This effect was due to a diminished p85 association to IRS-2 under pretreatment with TNF-{alpha} and insulin stimulation. However, IRS-1-associated PI 3-kinase activation in response to insulin was fully activated, as well as IRS-1 association to p85. These results correlated with those seen above on IRS-1 and IRS-2 phosphorylation in response to insulin in brown adipocytes pretreated with TNF-{alpha}, and provide further support to the functional differences between IRS-1 and IRS-2 in the insulin signaling pathways suggested above. Moreover, this selective impairment of IRS-2-associated PI 3-kinase signaling, but not IRS-1-associated PI 3-kinase signaling, may account for the partial inhibitory effect on glucose transport and also on adipogenic-related gene expression in response to insulin observed in brown adipocytes pretreated with TNF-{alpha}. In fact, we recently reported that the insulin effect on glucose transport and also on lipid synthesis were completely blunted in brown adipocytes pretreated with PI 3-kinase inhibitors (5). Whether or not IRS-2 could play a predominant role in glucose uptake and adipogenesis as opposed to IRS-1 in fetal brown adipocytes, merits further investigations.

In conclusion, our results show that TNF-{alpha} caused an impairment in the IRS-2-associated PI 3-kinase signaling in response to insulin, with IRS-1-associated PI 3-kinase signaling remaining unaltered. This effect led to an insulin resistance on glucose transport and also on the adipogenic-related gene expression in fetal brown adipocytes.


   Acknowledgments
 
We are grateful for valuable reagents provided by Drs. E. Rozengurt and J. Sinnet-Smith (Imperial Cancer Research Foundation, London) and Dr. M. White (Joslin Diabetes Center, Boston). We thank Dr. A. Alvarez for his expert technical assistance with the flow cytometer.


   Footnotes
 
1 This work was supported by a SAF 96/0115 grant from the Comision Interministerial de Ciencia y Tecnologia, Spain. Back

2 Equal contributors to the experimental work. Back

3 Recipient of a fellowship from the Comunidad Autonoma de Madrid, Spain. Back

4 Recipient of a fellowship from the Ministerio de Educacion y Ciencia. Back

Received October 10, 1997.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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