NGF Gene Expression and Secretion by Canine Adipocytes in Primary Culture: Upregulation by the Inflammatory Mediators LPS and TNFα

Abstract

Obesity is the commonest nutritional disorder of companion animals. In rodents and humans, white adipose tissue is a major endocrine and secretory organ, releasing adipokines linked to inflammation. In this study, we examined whether nerve growth factor (NGF), a target-derived neurotrophin central to the development/maintenance of sympathetic innervation and an inflammatory response protein, is synthesized and secreted by canine adipocytes. NGF mRNA was detected in each of the major fat depots (the subcutaneous, inguinal, gonadal, perirenal, and falciform ligaments) of dogs at similar levels. Canine adipocytes, differentiated from preadipocytes (inguinal depot) in primary culture, expressed the NGF gene and secreted NGF both pre- and post-differentiation. Treatment of the differentiated adipocytes with LPS resulted in a dramatic increase in NGF mRNA levels (20-fold at 24 h) and in NGF protein in the medium (60-fold at 24 h). The proinflammatory cytokine TNFα also led to a substantial increase in NGF mRNA levels (11-fold) and protein secretion (16-fold), while IL-6 had little effect. In contrast, dexamethasone decreased both NGF mRNA levels (80%) and protein release (60%). The PPARγ agonist rosiglitazone also reduced NGF secretion. These results demonstrate that canine white adipocytes synthesize and secrete NGF, the powerful upregulation by LPS and TNFα indicating that the neurotrophin is strongly linked to the inflammatory response in canine WAT. Canine adipocytes appear highly sensitive to inflammatory stimuli.

Key words

adipokines - adipose tissue - inflammation - mRNA - neurotrophin - preadipocytes

References

• 1 Mason E. Obesity in pet dogs.  Vet Rec. 1970;  86 612-616
• 2 Edney AT, Smith PM. Study of obesity in dogs visiting veterinary practices in the United Kingdom.  Vet Rec. 1986;  118 391-396
• 3 MacGreevy PD, Thomson PC, Pride C. et al . Prevalence of obesity in dogs examined by Australian veterinary practices and the risk factors involved.  Vet Rec. 2005;  156 695-702
• 4 Smith GK, Mayhew PD, Kapatkin AS. et al . Evaluation of risk factors for degenerative joint disease associated with hip dysplasia in German Shepherd Dogs, Golden Retrievers, Labrador Retrievers, and Rottweilers.  J Am Vet Med Assoc. 2001;  219 1719-1724
• 5 Rocchini AP, Marker P, Cervenka T. Time course of insulin resistance associated with feeding dogs a high-fat diet.  Am J Physiol Endocrinol Metab. 1997;  272 E147-E154
• 6 Truett AA, Borne AT, Monteiro MP, West DB. Composition of dietary fat affects blood pressure and insulin responses to dietary obesity in the dog.  Obesity Res. 1998;  6 137-146
• 7 Klinkenberg H, Sallander MH, Hedhammar A. Feeding, exercise, and weight identified as risk factors in canine diabetes mellitus.  J Nutr. 2006;  136 1985S-1987S
• 8 Kealy RD, Lawler DF, Ballam JM. et al . Effects of diet restriction on life span and age-related changes in dogs.  J Am Vet Med Assoc. 2002;  220 1315-1320
• 9 German AJ. The growing problem of obesity in dogs and cats.  J Nutr. 2006;  136 1940S-1946S
• 10 Trayhurn P, Beattie JH. Physiological role of adipose tissue: white adipose tissue as an endocrine and secretory organ.  Proc Nutr Soc. 2001;  60 329-339
• 11 Rajala MW, Scherer PE. Minireview: The adipocyte – at the crossroads of energy homeostasis, inflammation, and atherosclerosis.  Endocrinology. 2003;  144 3765-3773
• 12 Trayhurn P, Wood IS. Adipokines: Inflammation and the pleiotropic role of white adipose tissue.  Br J Nutr. 2004;  92 347-355
• 13 Kershaw EE, Flier JS. Adipose tissue as an endocrine organ.  J Clin Endocrinol Metab. 2004;  89 2548-2556
• 14 Sell H, Dietze-Schroeder D, Eckel J. The adipocyte-myocyte axis in insulin resistance.  Trends Endocrinol Metab. 2006;  17 416-422
• 15 Kahn SE, Hull RL, Utzschneider KM. Mechanisms linking obesity to insulin resistance and type 2 diabetes.  Nature. 2006;  444 840-846
• 16 Hotamisligil GS. Inflammation and metabolic disorders.  Nature. 2006;  444 860-867
• 17 Rayner DV, Trayhurn P. Regulation of leptin production: sympathetic nervous system interactions.  J Mol Med. 2001;  79 8-20
• 18 Levi-Montalcini R, Skaper SD, Dal Toso R, Petrelli L, Leon A. Nerve growth factor: from neurotrophin to neurokine.  Trends Neurosci. 1996;  19 514-520
• 19 Peeraully MR, Jenkins JR, Trayhurn P. NGF gene expression and secretion in white adipose tissue: regulation in 3T3-L1 adipocytes by hormones and inflammatory cytokines.  Am J Physiol Endocrinol Metab. 2004;  287 E331-E339
• 20 Eisele I, Wood IS, German AJ, Hunter L, Trayhurn P. Adipokine gene expression in dog adipose tissues and dog white adipocytes differentiated in primary culture.  Horm Metab Res. 2005;  37 474-481
• 21 Wang B, Jenkins JR, Trayhurn P. Expression and secretion of inflammation-related adipokines by human adipocytes differentiated in culture: Integrated response to TNF-α.  Am J Physiol Endocrinol Metab. 2005;  288 E731-E740
• 22 Barouch R, Kazimirsky G, Appel E, Brodie C. Nerve growth factor regulates TNF-α production in mouse macrophages via MAP kinase activation.  J Leukoc Biol. 2001;  69 1019-1026
• 23 Wang B, Trayhurn P. Acute and prolonged effects of TNF-α on the expression and secretion of inflammation-related adipokines by human adipocytes differentiated in culture.  Pflügers Archiv Eur J Physiol. 2006;  452 418-427
• 24 Wu P, Sato K, Yukawa S, Hikasa Y, Kagota K. Differentiation of stromal-vascular cells isolated from canine adipose tissues in primary culture.  J Vet Med Sci. 2001;  63 17-23
• 25 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCT method.  Methods. 2001;  25 402-408
• 26 Bulló M, Peeraully MR, Trayhurn P. Stimulation of NGF expression and secretion in 3T3-L1 adipocytes by prostaglandins PGD₂, PGJ₂, and Δ¹²-PGJ₂.  Am J Physiol Endocrinol Metab. 2005;  289 E62-E67
• 27 Champigny O, Ricquier D, Blondel O. et al . β₃-adrenergic receptor stimulation restores message and expression of brown-fat mitochondrial uncoupling protein in adult dogs.  Proc Natl Acad Sci USA. 1991;  88 10774-10777
• 28 Freund V, Pons F, Joly V. et al . Upregulation of nerve growth factor expression by human airway smooth muscle cells in inflammatory conditions.  Eur Respir J. 2002;  20 458-463
• 29 Toyomoto M, Ohta M, Okumura K. et al . Prostaglandins are powerful inducers of NGF and BDNF production in mouse astrocyte cultures.  FEBS Lett. 2004;  562 211-215
• 30 Peeraully MR, Sievert H, Bullo M, Wang B, Trayhurn P. Prostaglandin D₂ and J₂-series (PGJ₂, Δ¹²-PGJ₂) prostaglandins stimulate IL-6 and MCP-1, but inhibit leptin, expression and secretion by 3T3-L1 adipocytes.  Pflügers Archiv Eur J Physiol. 2006;  453 177-187
• 31 Lin Y, Lee H, Berg AH. et al . The lipopolysaccharide-activated toll-like receptor (TLR)-4 induces synthesis of the closely related receptor TLR-2 in adipocytes.  J Biol Chem. 2000;  275 24255-24263
• 32 Yoshida T, Hayashi M, Monkawa T, Saruta T. Regulation of obese mRNA expression by hormonal factors in primary cultures of rat adipocytes.  Eur J Endocrinol. 1996;  135 619-625
• 33 Miell JP, Englaro P, Blum WF. Dexamethasone induces an acute and sustained rise in circulating leptin levels in normal human subjects.  Horm Metab Res. 1996;  28 704-707
• 34 Mitchell SE, Rees WD, Hardie LJ. et al . ob gene expression and secretion of leptin following differentiation of rat preadipocytes to adipocytes in primary culture.  Biochem Biophys Res Commun. 1997;  230 360-364
• 35 Ishioka K, Soliman MM, Honjoh T. et al . Dexamethasone increases serum leptin concentration in dogs.  Vet J. 2002;  164 295-297
• 36 Shojima N, Sakoda H, Ogihara T. et al . Humoral regulation of resistin expression in 3T3-L1 and mouse adipose cells.  Diabetes. 2002;  51 1737-1744
• 37 Oller do Nascimento C, Hunter L, Trayhurn P. Regulation of haptoglobin gene expression in 3T3-L1 adipocytes by cytokines, catecholamines, and PPARγ.  Biochem Biophys Res Commun. 2004;  313 702-708
• 38 Staels B, Fruchart J-C. Therapeutic roles of peroxisome proliferator-activated receptor agonists.  Diabetes. 2005;  54 2460-2470
• 39 Ghanim H, Dhindsa S, Aljada A. et al . Low-dose rosiglitazone exerts an antiinflammatory effect with an increase in adiponectin independently of free fatty acid fall and insulin sensitization in obese Type 2 diabetics.  J Clin Endocrinol Metab. 2006;  91 3553-3558
• 40 Sharma AM, Staels B. Peroxisome proliferator-activated receptor γ and adipose tissue: understanding obesity-related changes in regulation of lipid and glucose metabolism.  J Clin Endocrinol Metab. 2007;  92 386-395
• 41 De Vos P, Lefebvre AM, Miller SG. et al . Thiazolidinediones repress ob gene expression in rodents via activation of peroxisome proliferator-activated receptor γ.  J Clin Invest. 1996;  98 1004-1009
• 42 Kallen CB, Lazar MA. Antidiabetic thiazolidinediones inhibit leptin (ob) gene expression in 3T3-L1 adipocytes.  Proc Natl Acad Sci USA. 1996;  93 5793-5796
• 43 Steppan CM, Bailey ST, Bhat S. et al . The hormone resistin links obesity to diabetes.  Nature. 2001;  409 307-312
• 44 Moore GB, Chapman H, Holder JC. et al . Differential regulation of adipocytokine mRNAs by rosiglitazone in db/db mice.  Biochem Biophys Res Commun. 2001;  286 735-741
• 45 Weisberg SP, MacCann D, Desai M. et al . Obesity is associated with macrophage accumulation in adipose tissue.  J Clin Invest. 2003;  112 1796-1808
• 46 Xu H, Barnes GT, Yang Q. et al . Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance.  J Clin Invest. 2003;  112 1821-1830
• 47 Kirkness EF, Bafna V, Halpern AL. et al . The dog genome: survey sequencing and comparative analysis.  Science. 2003;  301 1898-1903
• 48 Lindblad-Toh K, Wade CM, Mikkelsen TS. et al . Genome sequence, comparative analysis and haplotype structure of the domestic dog.  Nature. 2005;  438 803-819
• 49 Bergman RN, Citters GW Van, Mittelman SD. et al . Central role of the adipocyte in the metabolic syndrome.  J Investig Med. 2001;  49 119-126

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