Aging-Induced Brain-Derived Neurotrophic Factor in Adipocyte Progenitors Contributes to Adipose Tissue Dysfunction
Hyun-Doo Song1#, Sang Nam Kim2#, Abhirup Saha2#, Sang-Yeop Ahn1, Seun Akindehin1, Yeonho Son2, Yoon Keun Cho2, MinSu Kim2, Ji-Hyun Park2, Young-Suk Jung3, Yun-Hee Lee2,*
1College of Pharmacy, Yonsei University, Incheon, Republic of Korea. 2College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Republic of Korea. 3College of Pharmacy, Pusan National University, Busan, Republic of Korea.
Aging-related adipose tissue dysfunction contributes to the progression of chronic metabolic diseases. We investigated the role of age-dependent expression of a neurotrophin, brain-derived neurotrophic factor (BDNF) in adipose tissue. Pro-BDNF expression was elevated in epididymal white adipose tissue (eWAT) with advanced age, which was associated with the reduction in sympathetic innervation. Interestingly, BDNF expression was enriched in PDGFRα+ adipocyte progenitors isolated from eWAT, with age-dependent increase in expression. In vitro pro-BDNF treatment caused apoptosis in adipocytes differentiated from C3H10T1/2 cells, and siRNA knockdown of sortilin mitigated these effects. Tamoxifen-inducible PDGFRα+ cell-specific deletion of BDNF (BDNFPdgfra KO) reduced pro-BDNF expression in eWAT, prevented age-associated declines in sympathetic innervation and mitochondrial content in eWAT, and improved insulin sensitivity. Moreover, BDNFPdgfra KO mice showed reduced expression of aging-induced inflammation and senescence markers in eWAT. Collectively, these results identified the upregulation of pro-BDNF expression in adipocyte progenitors as a feature of visceral white adipose tissue aging and suggested that inhibition of BDNF expression in adipocyte progenitors is potentially beneficial to prevent aging-related adipose tissue dysfunction.
Hyun-Doo Song,Sang Nam Kim,Abhirup Saha, et al. Aging-Induced Brain-Derived Neurotrophic Factor in Adipocyte Progenitors Contributes to Adipose Tissue Dysfunction[J]. Aging and disease,
Figure 1. BDNF expression was upregulated in epididymal adipose tissue with advanced age, but not in brown adipose tissue and inguinal white adipose tissue. (A-D) Immunoblot analysis of BDNF and senescence marker expression in supra-scapular brown adipose tissue (BAT), epididymal white adipose tissue (eWAT), and inguinal white adipose tissue (iWAT) of mice at the indicated ages. N= 4, mean ± S.E.M, p value was calculated by t-test (Full length blots in supplementary Fig. 2).
Figure 2. Age-dependent increase in pro-BDNF expression was associated with reduction in sympathetic innervation and mitochondrial activity in eWAT. (A) Immunoblot analysis of BDNF, TH and p-HSL expression in epididymal white adipose tissue (eWAT) of mice at the indicated ages. (n = 4, mean ± S.E.M, **p<0.01, ***p<0.001). (B, C) quantitative PCR analysis. (n = 4, mean ± S.E.M, *p<0.05, **p<0.01, ***p<0.001). (D) Analysis of oxygen consumption rate (OCR) of eWAT obtained from 2, 12 and 18-month old mice with a series of treatments of indicated drugs (oligomycin, carbonyl cyanide-4(trifluoromethoxy)phenylhydrazone (FCCP), and potassium cyanide (KCN)) (n = 3, mean ± S.E.M, *p<0.05, **p<0.01, ***p<0.001). (E) MitoTracker Red CMXRos staining in adipocytes differentiated from PDGFRα+ cells of eWAT of 2, 12, and 18-month-old mice.
Figure 3. Upregulation of pro-BDNF expression by 12 weeks of HFD feeding (A) Immunoblot analysis of BDNF, TH and p-HSL expression in epididymal white adipose tissue (eWAT) mice fed with high fat diet and chow diet for 12 weeks. (n = 6, mean ± S.E.M, *p<0.05, **p<0.01).
Figure 4. The major cellular source of pro-BDNF expression in eWAT is PDGFRα+ adipocyte progenitors. (A) quantitative PCR analysis of Bdnf expression in adipocytes and PDGFRA+ cells isolated from eWAT of mice at the indicated ages (n = 3, mean ± S.E.M, ***p<0.001). Plin1 and Pdgfra expressions were used as specific markers for adipocytes and progenitor cells, respectively. (B) Immunoblot analysis of BDNF expression in adipocytes and PDGFRA+ cells isolated from eWAT of mice at the indicated ages (n = 3, mean ± S.E.M, ***p<0.001). Full images of Western blots are shown in supplementary Fig. 4.
Figure 5. proBDNF treatment-induced apoptosis/necroptosis of adipocytes required sortilin expression. (A) Immunoblot analysis of sortilin expression and apoptosis/necroptosis markers in adipocytes differentiated from C3H10T1/2 cells. (B) Immunoblot analysis of sortilin in adipocytes differentiated from C3H10T1/2 treated with siRNA or scrambled sequence controls (negative controls) (mean ± SEM; n = 4, *** p < 0.001). (C) Immunoblot analysis of cell surface protein detection in adipocytes differentiated from C3H10T1/2 cells treated with vehicle or pro-BDNF (10ng/ml) for 24 h (n = 4, means ± SEM, *** p < 0.001). Full images of Western blots are shown in supplementary Fig. 5.
Figure 6. PDGFRα+ cell-specific KO reduced inflammatory and senescence marker expression in eWAT and insulin resistance of mice with advanced age. (A) Immunoblot analysis of BDNF and TH expression in epididymal white adipose tissue (eWAT) of BDNFpdgfra KO and WT mice at the indicated ages. (n = 5 per condition, mean ± S.E.M, *p<0.05, **p<0.01, *** p < 0.001). (B) Quantitative PCR analysis of eWAT of BDNFpdgfra KO and WT mice at the indicated ages. (n = 5, mean ± S.E.M, *p<0.05, **p<0.01, ***p<0.001). (C) Immunoblot analysis of mitochondrial makers involved in mitochondrial oxidative phosphorylation. (D) BODIPY staining of adipocytes differentiated from PDGFRα+ cells that were isolated from eWAT of BDNFpdgfra KO and WT mice (n = 4, mean ± S.E.M, **p<0.01). (E) Measurement of glucose tolerance test (GTT) in WT and BDNFpdgfraKO mice and the area under the curve of GTT plots. N = 5, mean ± S.E.M, **p<0.01. Full images of Western blots are shown in supplementary Fig. 6.
Figure 7. PDGFRα+ cell-specific KO reduced apoptosis and necroptosis in eWAT of mice with advanced age. (A) Quantitative PCR analysis of eWAT of BDNFpdgfra KO and WT mice at the indicated ages. (n = 5, mean ± S.E.M, *p<0.05, **p<0.01, ***p<0.001). (B) Immunostaining of F4/80 in paraffin sections of eWAT of BDNFpdgfra KO and WT mice. DAPI was used as a nuclear counterstain. (C) Immunoblot analysis of apoptosis/necroptosis makers in eWAT of BDNFpdgfra KO and WT mice. (D) Immunoblot analysis of sortilin expression in plasma membrane fractions of eWAT of WT and BDNFpdgfra KO mice (n = 5, means ± SEM). Full images of Western blots are shown in supplementary Fig. 7.
Houtkooper RH, Argmann C, Houten SM, Canto C, Jeninga EH, Andreux PA, et al. (2011). The metabolic footprint of aging in mice. Sci Rep, 1:134.
Stout MB, Tchkonia T, Pirtskhalava T, Palmer AK, List EO, Berryman DE, et al. (2014). Growth hormone action predicts age-related white adipose tissue dysfunction and senescent cell burden in mice. Aging (Albany NY), 6:575-586.
Villaret A, Galitzky J, Decaunes P, Esteve D, Marques MA, Sengenes C, et al. (2010). Adipose tissue endothelial cells from obese human subjects: differences among depots in angiogenic, metabolic, and inflammatory gene expression and cellular senescence. Diabetes, 59:2755-2763.
Bahler L, Verberne HJ, Admiraal WM, Stok WJ, Soeters MR, Hoekstra JB, et al. (2016). Differences in Sympathetic Nervous Stimulation of Brown Adipose Tissue Between the Young and Old, and the Lean and Obese. Journal of Nuclear Medicine, 57:372-377.
Martyniak K, Masternak MM (2017). Changes in adipose tissue cellular composition during obesity and aging as a cause of metabolic dysregulation. Exp Gerontol, 94:59-63.
Tchkonia T, Morbeck DE, Von Zglinicki T, Van Deursen J, Lustgarten J, Scrable H, et al. (2010). Fat tissue, aging, and cellular senescence. Aging Cell, 9:667-684.
Schosserer M, Grillari J, Wolfrum C, Scheideler M (2018). Age-Induced Changes in White, Brite, and Brown Adipose Depots: A Mini-Review. Gerontology, 64:229-236.
Baker DJ, Wijshake T, Tchkonia T, LeBrasseur NK, Childs BG, van de Sluis B, et al. (2011). Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature, 479:232-236.
Nisoli E, Tonello C, Carruba MO (1998). Nerve growth factor, beta3-adrenoceptor and uncoupling protein 1 expression in rat brown fat during postnatal development. Neurosci Lett, 246:5-8.
Fischer AW, Schlein C, Cannon B, Heeren J, Nedergaard J (2018). Intact innervation is essential for diet-induced recruitment of brown adipose tissue. Am J Physiol Endocrinol Metab.
Festuccia WT, Blanchard PG, Richard D, Deshaies Y (2010). Basal adrenergic tone is required for maximal stimulation of rat brown adipose tissue UCP1 expression by chronic PPAR-gamma activation. Am J Physiol Regul Integr Comp Physiol, 299:R159-167.
Kowianski P, Lietzau G, Czuba E, Waskow M, Steliga A, Morys J (2018). BDNF: A Key Factor with Multipotent Impact on Brain Signaling and Synaptic Plasticity. Cell Mol Neurobiol, 38:579-593.
Ernfors P, Lee KF, Jaenisch R (1994). Mice lacking brain-derived neurotrophic factor develop with sensory deficits. Nature, 368:147-150.
Fundin BT, Silos-Santiago I, Ernfors P, Fagan AM, Aldskogius H, DeChiara TM, et al. (1997). Differential dependency of cutaneous mechanoreceptors on neurotrophins, trk receptors, and P75 LNGFR. Dev Biol, 190:94-116.
Rice FL, Albers KM, Davis BM, Silos-Santiago I, Wilkinson GA, LeMaster AM, et al. (1998). Differential dependency of unmyelinated and A delta epidermal and upper dermal innervation on neurotrophins, trk receptors, and p75LNGFR. Dev Biol, 198:57-81.
Biddinger JE, Fox EA (2014). Reduced Intestinal Brain-Derived Neurotrophic Factor Increases Vagal Sensory Innervation of the Intestine and Enhances Satiation. The Journal of Neuroscience, 34:10379-10393.
Mowla SJ, Farhadi HF, Pareek S, Atwal JK, Morris SJ, Seidah NG, et al. (2001). Biosynthesis and post-translational processing of the precursor to brain-derived neurotrophic factor. J Biol Chem, 276:12660-12666.
Sun Y, Lim Y, Li F, Liu S, Lu JJ, Haberberger R, et al. (2012). ProBDNF collapses neurite outgrowth of primary neurons by activating RhoA. PLoS One, 7:e35883.
Teng HK, Teng KK, Lee R, Wright S, Tevar S, Almeida RD, et al. (2005). ProBDNF induces neuronal apoptosis via activation of a receptor complex of p75NTR and sortilin. J Neurosci, 25:5455-5463.
Lee YH, Petkova AP, Mottillo EP, Granneman JG (2012). In vivo identification of bipotential adipocyte progenitors recruited by beta3-adrenoceptor activation and high-fat feeding. Cell Metab, 15:480-491.
Kim SN, Kwon HJ, Im SW, Son YH, Akindehin S, Jung YS, et al. (2017). Connexin 43 is required for the maintenance of mitochondrial integrity in brown adipose tissue. Sci Rep, 7:7159.
el Bouazzaoui F, Henneman P, Thijssen P, Visser A, Koning F, Lips MA, et al. (2014). Adipocyte telomere length associates negatively with adipocyte size, whereas adipose tissue telomere length associates negatively with the extent of fibrosis in severely obese women. Int J Obes (Lond), 38:746-749.
Lee YH, Petkova AP, Konkar AA, Granneman JG (2015). Cellular origins of cold-induced brown adipocytes in adult mice. Faseb j, 29:286-299.
Lee YH, Kim SN, Kwon HJ, Maddipati KR, Granneman JG (2016). Adipogenic role of alternatively activated macrophages in beta-adrenergic remodeling of white adipose tissue. Am J Physiol Regul Integr Comp Physiol, 310:R55-65.
Lee YH, Kim SN, Kwon HJ, Granneman JG (2017). Metabolic heterogeneity of activated beige/brite adipocytes in inguinal adipose tissue. Sci Rep, 7:39794.
Nykjaer A, Lee R, Teng KK, Jansen P, Madsen P, Nielsen MS, et al. (2004). Sortilin is essential for proNGF-induced neuronal cell death. Nature, 427:843-848.
Howard L, Wyatt S, Nagappan G, Davies AM (2013). ProNGF promotes neurite growth from a subset of NGF-dependent neurons by a p75NTR-dependent mechanism. Development, 140:2108-2117.
Dieni S, Matsumoto T, Dekkers M, Rauskolb S, Ionescu MS, Deogracias R, et al. (2012). BDNF and its pro-peptide are stored in presynaptic dense core vesicles in brain neurons. J Cell Biol, 196:775-788.
Mizui T, Ohira K, Kojima M (2017). BDNF pro-peptide: a novel synaptic modulator generated as an N-terminal fragment from the BDNF precursor by proteolytic processing. Neural Regen Res, 12:1024-1027.
Marosi K, Mattson MP (2014). BDNF mediates adaptive brain and body responses to energetic challenges. Trends Endocrinol Metab, 25:89-98.
Tsuchida A, Nonomura T, Ono-Kishino M, Nakagawa T, Taiji M, Noguchi H (2001). Acute effects of brain-derived neurotrophic factor on energy expenditure in obese diabetic mice. International Journal Of Obesity, 25:1286.
Tsuchida A, Nakagawa T, Itakura Y, Ichihara J, Ogawa W, Kasuga M, et al. (2001). The effects of brain-derived neurotrophic factor on insulin signal transduction in the liver of diabetic mice. Diabetologia, 44:555-566.
Nakagawa T, Tsuchida A, Itakura Y, Nonomura T, Ono M, Hirota F, et al. (2000). Brain-derived neurotrophic factor regulates glucose metabolism by modulating energy balance in diabetic mice. Diabetes, 49:436-444.
Shetty AK, Kodali M, Upadhya R, Madhu LN (2018). Emerging Anti-Aging Strategies - Scientific Basis and Efficacy. Aging Dis, 9:1165-1184.
Choi SH, Bylykbashi E, Chatila ZK, Lee SW, Pulli B, Clemenson GD, et al. (2018). Combined adult neurogenesis and BDNF mimic exercise effects on cognition in an Alzheimer's mouse model. Science, 361.
Nykjaer A, Willnow TE (2012). Sortilin: a receptor to regulate neuronal viability and function. Trends Neurosci, 35:261-270.
Rabinowich L, Fishman S, Hubel E, Thurm T, Park WJ, Pewzner-Jung Y, et al. (2015). Sortilin deficiency improves the metabolic phenotype and reduces hepatic steatosis of mice subjected to diet-induced obesity. J Hepatol, 62:175-181.
Robin Gourmelon, Sandrine Donadio-Andréi, Karim Chikh, Muriel Rabilloud, Elisabetta Kuczewski, Anne-Sophie Gauchez, Anne Charrié, Pierre-Yves Brard, Raphaëlle Andréani, Jean-Cyril Bourre, Christine Waterlot, Domitille Guédel, Anne Mayer, Emmanuel Disse, Charles Thivolet, Hélène Du Boullay, Claire Falandry, Thomas Gilbert, Anne François-Joubert, Antoine Vignoles, Catherine Ronin, Marc Bonnefoy. Subclinical Hypothyroidism: is it Really Subclinical with Aging?[J]. Aging and disease, 2019, 10(3): 520-529.