Adiponectin is a circulating factor that is secreted from adipocytes (Hu et al., 1996; Maeda et al., 1996; Nakano et al., 1996; Scherer et al., 1995). Three types of receptors have been identified for this uniquely abundant circulating protein: AdipoRs, calreticulin, and T-cadherin.

AdipoR1 was discovered by expression cloning from a C2C12 myotube cDNA library through evaluations of the binding of biotin-labeled globular adiponectin produced in E. coli as bait (Yamauchi et al., 2003). It belongs to the PAQR receptor family, which has a 7-transmembrane domain with an opposite topology to the GPCR family (Tang et al., 2005).

Calreticulin is an endoplasmic reticulum (ER) luminal Ca2+-buffering chaperone that exists in the ER of cells (Mendlov and Conconi, 2010; Michalak et al., 2009). Cell surface exposure to calreticulin was initially reported to initiate the clearance of viable or apoptotic cells through binding to LRP on phagocytes (Gardai et al., 2005). A subsequent study demonstrated that adiponectin opsonized apoptotic cells, and the phagocytosis of cell corpses was mediated by the binding of adiponectin expressed in insect cells or E. coli to calreticulin on the macrophage cell surface (Takemura et al., 2007).

T-Cadherin was discovered by expression cloning from a C2C12 myotube cDNA library through evaluations of cell binding to coated recombinant adiponectin produced in HEK293 mammalian cells (Hug et al., 2004). It is classified as a member of the classical cadherins, such as E-cadherin and N-cadherin, due to its high homology of five extracellular cadherin repeats (Hulpiau and van Roy, 2009). However, T-cadherin is a unique cadherin with a glycosylphosphatidylinositol (GPI) anchor on its C terminus and does not possess the transmembrane or intracellular domain generally required for signaling, which may hinder the function of T-cadherin as an adiponectin receptor.

The adiponectin protein accumulates in tissues, such as the heart, muscle, and vascular endothelium, through binding with T-cadherin (Denzel et al., 2010; Fujishima et al., 2017; Matsuda et al., 2015; Parker-Duffen et al., 2013; Tanaka et al., 2019). In T-cadherin null mice, the accumulation of the adiponectin protein was completely absent in these tissues, and, thus, HMW multimer adiponectin accumulated in blood (Denzel et al., 2010; Matsuda et al., 2015; Parker-Duffen et al., 2013). These findings were in contrast to the lack of significant changes in plasma adiponectin levels in AdipoR1- and R2-double knockout mice (Yamauchi et al., 2007). Human SNP studies including GWAS also indicated the importance of T-cadherin, but not AdipoRs or calreticulin, for plasma adiponectin levels, cardiovascular diseases, and glucose homeostasis (Buniello et al., 2019; Chung et al., 2011; Dastani et al., 2012; Kitamoto et al., 2016; Morisaki et al., 2012).

Numerous studies have attributed the functions of adiponectin to either of these receptors by showing a decrease in their functions via the genetic loss or mRNA knockdown of their receptors, including AdipoRs (Straub and Scherer, 2019; Yamauchi et al., 2014; Yamauchi et al., 2003; Yamauchi et al., 2007), calreticulin (Takemura et al., 2007), and T-cadherin (Denzel et al., 2010; Fujishima et al., 2017; Parker-Duffen et al., 2013; Tanaka et al., 2019). However, the direct binding of native adiponectin in biological fluids, such as serum, to its receptor warrants further study.

We herein demonstrated that native adiponectin in serum bound to cells expressing T-cadherin, but not to those expressing AdipoRs or calreticulin.

We investigated the binding of native adiponectin in serum to three adiponectin receptors by transiently overexpressing the cDNA of each receptor in HEK293 cells (Figure 1A). We directly examined mouse serum as the ligand solution, including the most native adiponectin, purified adiponectin from mouse serum (Fukuda et al., 2017), and full-length recombinant adiponectin produced in HEK293 cells. Native-PAGE showed differences in the distribution of molecular species between serum or purified adiponectin and recombinant adiponectin (Figure 1B). Recombinant adiponectin contained a lower amount of HMW multimer adiponectin than mouse serum and purified adiponectin from serum (Figure 1B). Transient transfection resulted in the successful overexpression of each receptor based on their expression levels quantified by RT-qPCR (Figure 1C). The treatment of cells with different preparations of adiponectin at 4°C for 1 hr resulted in the binding of prepared adiponectin only to cells expressing mouse T-cadherin (Figure 1D). Mouse serum and purified adiponectin showed similar binding, whereas recombinant adiponectin containing a lower amount of 6-mer and the HMW multimer exhibited markedly weaker binding (Figure 1D). The results of a native-PAGE analysis showed that more than 6-mer of multimeric adiponectin specifically bound to cells expressing mouse T-cadherin (Figure 1—figure supplement 1), which is consistent with previous findings (Fukuda et al., 2017; Hug et al., 2004). The dose-response study revealed the specific and saturable binding of native adiponectin in serum to cells expressing T-cadherin (Figure 1E).

Figure 1 with 2 supplements see all

Download asset Open asset

Similar results were obtained when human cDNAs were overexpressed and the binding of adiponectin in human serum was assessed (Figure 1—figure supplement 2A–D). We previously reported that purified recombinant T-cadherin bound purified adiponectin with an affinity of KD = 1.0 nM (Fukuda et al., 2017). The present results showed the saturable binding of native adiponectin in serum to cells expressing T-cadherin, which is consistent with previous findings (Fukuda et al., 2017). Regarding AdipoR1 and calreticulin, three possibilities have been proposed: they were not effectively translated, were not effectively presented on the cell surface, or did not support the binding of native adiponectin in serum to cells.

To confirm that all receptors were effectively translated and presented on the cell surface, we expressed affinity-tagged receptors in HEK293 cells (Figure 2A). We added a high-affinity PA tag (Fujii et al., 2014) to the N termini of T-cadherin (Ciatto et al., 2010) and calreticulin (Mendlov and Conconi, 2010) and to the C terminus of AdipoR1 (Yamauchi et al., 2014) such that each receptor exposed the PA tag outside of the cell. Transiently expressing cells were surface-biotinylated, and lysates were applied to streptavidin beads. Total cell lysates (Figure 2B Total) and streptavidin-captured cell-surface proteins (Figure 2B Cell surface) were analyzed by Western blotting. The anti-PA-tag antibody NZ-1 detected similar levels of all receptors in total cell lysates and cell surface fractions, indicating that these receptors were successfully translated and expressed on the cell surface. Although AdipoR1 poorly migrated on the SDS-PAGE gel, this may have been due to heat-induced protein crosslinking or aggregate formation during sample processing (Tanford and Reynolds, 1976). The correct sorting of this protein to the cell surface suggested that AdipoR1 was expressed with the correct conformation on the cell surface. Under these conditions, a binding study with mouse serum revealed the dose-dependent binding of native adiponectin to cells expressing PA-tagged T-cadherin, but not to those expressing PA-tagged calreticulin or AdipoR1 (Figure 2C). Taken together with the results of the overexpression study (Figure 1), native adiponectin in serum bound to cells expressing T-cadherin, but not those expressing calreticulin or AdipoR1.

Figure 2

Download asset Open asset

We then generated stably expressing CHO cells (Figure 2D). The successful expression of the receptors in these cells was confirmed by Western blotting (Figure 2E). Serum adiponectin binding was only observed on cells expressing PA-tagged T-cadherin (Figure 2F). In contrast, the binding of the PA tag antibody NZ-1 to intact cells was detected on cells expressing PA-tagged T-cadherin and AdipoR1 (Figure 2G). These results demonstrated that AdipoR1 was stably expressed on the cell surface with the expected topology of the PA tag outside of cells, but did not induce the binding of native adiponectin in serum. Calreticulin was not recognized by NZ-1 in the binding study (Figure 2G), suggesting that the PA tag at the N terminus of calreticulin was not accessible by NZ-1.

This may have been due to some steric hindrance because calreticulin with the N-terminal PA tag was detected on Western blots (Figure 2E) and immunostaining of fixed and permeabilized cells by NZ-1 (Figure 2H). Calreticulin is essentially an ER-resident protein (Gardai et al., 2005; Takemura et al., 2007). Therefore, stably expressed calreticulin in CHO cells may not have been sorted to the cell surface.

Based on these different approaches, we concluded that the expression of AdipoR1 may not promote native adiponectin binding. We also concluded that if calreticulin is expressed on the cell surface, it may not promote native adiponectin binding. The present results demonstrated that only the expression of T-cadherin on the cell surface may increase the binding of native adiponectin.

Since the above studies employed artificial expression systems, and AdipoR1 and T-cadherin were both identified from the C2C12 cDNA library (Hug et al., 2004; Yamauchi et al., 2003), we examined native adiponectin binding to C2C12 myotubes (Figure 3A). The absolute expression level of T-cadherin mRNA in differentiated C2C12 myotubes was markedly higher than that of AdipoRs (Figure 3B). We investigated the knockdown effects of these receptors on the binding of serum-containing adiponectin in C2C12 myotubes (Figure 3C,D). The introduction of RNAi before differentiation resulted in the effective knockdown of T-cadherin, AdipoR1, or AdipoR1 and R2 after 3 days of differentiation (Figure 3C). The knockdown of T-cadherin resulted in significant reductions in adiponectin binding (Figure 3D,E). The knockdown of AdipoR1 or both AdipoR1 and R2 did not significantly reduce adiponectin binding (Figure 3D,E). Although slight decreases were observed in adiponectin binding by the knockdown of AdipoR1 or both AdipoR1 and R2, the strong correlation (R² = 0.9896) between T-cadherin expression and adiponectin binding at all experimental points suggested that these changes were due to the decreased expression of T-cadherin (Figure 3F,G). Collectively, these results indicated that native adiponectin binding also depends on the amount of T-cadherin expressed in C2C12 myotubes.

Figure 3

Download asset Open asset

Here, we simultaneously compared three adiponectin receptors. The expression of T-cadherin gave adiponectin binding, which is consistent with our previous finding showing that recombinant T-cadherin binds HMW adiponectin in a 1: one ratio with high affinity (Fukuda et al., 2017). There was no detectable binding of adiponectin on the AdipoR or calreticulin. AdipoR was discovered by expression cloning. The overexpression of AdipoR was expected to promote ligand binding. The initial discovery of AdipoRs also indicated that HEK293 cells overexpressing AdipoRs bound E. coli recombinant globular adiponectin (Yamauchi et al., 2003). Therefore, difficulties are associated with speculating about the much weaker affinity or the requirement for some ‘accessory’ proteins to confer adiponectin binding activity to AdipoRs.

Since numerous studies indicated that AdipoRs mediate adiponectin signaling in a number of cell types, an additional activating mechanism for AdipoRs by adiponectin, that is, the reductive or proteolytic generation of the trimer, monomer, and/or globular adiponectin, may exist. The results of the present study, which focused on the direct binding of native HMW adiponectin, may indicate the activation of AdipoRs by low-molecular-weight (LMW) forms. On the other hand, in the case of calreticulin, this was evidenced by a neutralizing antibody treatment inhibiting the adiponectin interaction with cells (Takemura et al., 2007). Therefore, some ‘accessory’ proteins may be required to confer adiponectin binding activity to calreticulin.

T-cadherin binds clinically important HMW multimer adiponectin with high affinity (Fukuda et al., 2017) and mediates adiponectin-induced exosome biogenesis and ceramide efflux to exosomes (Obata et al., 2018). Such exosome-effect required T-cadherin, but not AdipoRs. The exosome mediates cell-cell communication by transferring signaling components such as microRNAs, bioactive lipids, and proteins in addition to its role in waste disposal (Kita et al., 2019; van Niel et al., 2018). The stimulation of exosome biogenesis by adiponectin was the first demonstration of a secreted factor modulating exosome biogenesis and secretion (Obata et al., 2018). Adiponectin in serum or purified native adiponectin together with T-cadherin accumulated inside multivesicular bodies, the site of exosome generation, both in cultured endothelial cells and the in vivo wild-type mouse aorta (Obata et al., 2018). The systemic level of exosomes in blood was decreased by approximately 50% following the genetic loss of adiponectin or T-cadherin, but was increased by the overexpression of adiponectin in mice (Obata et al., 2018). The molecular mechanisms by which adiponectin stimulates exosome biogenesis are currently under investigation. We speculate that native HMW adiponectin with its multimeric structure may cause the higher-order clustering of T-cadherin, a membrane-anchored protein that resides in lipid rafts, and, thus, may stimulate exosome biogenesis. Adiponectin-induced increases in exosome biogenesis were not restricted to cultured endothelial cells because they were also observed in C2C12-differentiated myotubes (Tanaka et al., 2019) These findings support T-cadherin mediating adiponectin functions as a receptor for native HMW multimer adiponectin (Kita et al., 2019). The present results may further contribute to clarifying the activating mechanism of AdipoRs by LMW adiponectin, generated by reduction or proteolytic cleavage, followed by HMW adiponectin binding to cell surface T-cadherin.

All data were deposited in Dryad at https://doi.org/10.5061/dryad.82557c0.

1. 1. Kita S
   2. Fukuda S
   3. Maeda N
   4. Shimomura I

   (2019) Dryad Digital Repository

   Data from: Native adiponectin in serum binds to cells expressing T-cadherin, but not AdipoRs or calreticulin.

   https://doi.org/10.5061/dryad.82557c0

1. 1. Buniello A
   2. MacArthur JAL
   3. Cerezo M
   4. Harris LW
   5. Hayhurst J
   6. Malangone C
   7. McMahon A
   8. Morales J
   9. Mountjoy E
   10. Sollis E
   11. Suveges D
   12. Vrousgou O
   13. Whetzel PL
   14. Amode R
   15. Guillen JA
   16. Riat HS
   17. Trevanion SJ
   18. Hall P
   19. Junkins H
   20. Flicek P
   21. Burdett T
   22. Hindorff LA
   23. Cunningham F
   24. Parkinson H

   (2019) The NHGRI-EBI GWAS catalog of published genome-wide association studies, targeted arrays and summary statistics 2019

   Nucleic Acids Research 47:D1005–D1012.

   https://doi.org/10.1093/nar/gky1120

   • PubMed
   • Google Scholar
2. 1. Chung CM
   2. Lin TH
   3. Chen JW
   4. Leu HB
   5. Yang HC
   6. Ho HY
   7. Ting CT
   8. Sheu SH
   9. Tsai WC
   10. Chen JH
   11. Lin SJ
   12. Chen YT
   13. Pan WH

   (2011) A genome-wide association study reveals a quantitative trait locus of adiponectin on CDH13 that predicts cardiometabolic outcomes

   Diabetes 60:2417–2423.

   https://doi.org/10.2337/db10-1321

   • PubMed
   • Google Scholar
3. 1. Ciatto C
   2. Bahna F
   3. Zampieri N
   4. VanSteenhouse HC
   5. Katsamba PS
   6. Ahlsen G
   7. Harrison OJ
   8. Brasch J
   9. Jin X
   10. Posy S
   11. Vendome J
   12. Ranscht B
   13. Jessell TM
   14. Honig B
   15. Shapiro L

   (2010) T-cadherin structures reveal a novel adhesive binding mechanism

   Nature Structural & Molecular Biology 17:339–347.

   https://doi.org/10.1038/nsmb.1781

   • PubMed
   • Google Scholar
4. 1. Dastani Z
   2. Hivert MF
   3. Timpson N
   4. Perry JR
   5. Yuan X
   6. Scott RA
   7. Henneman P
   8. Heid IM
   9. Kizer JR
   10. Lyytikäinen LP
   11. Fuchsberger C
   12. Tanaka T
   13. Morris AP
   14. Small K
   15. Isaacs A
   16. Beekman M
   17. Coassin S
   18. Lohman K
   19. Qi L
   20. Kanoni S
   21. Pankow JS
   22. Uh HW
   23. Wu Y
   24. Bidulescu A
   25. Rasmussen-Torvik LJ
   26. Greenwood CM
   27. Ladouceur M
   28. Grimsby J
   29. Manning AK
   30. Liu CT
   31. Kooner J
   32. Mooser VE
   33. Vollenweider P
   34. Kapur KA
   35. Chambers J
   36. Wareham NJ
   37. Langenberg C
   38. Frants R
   39. Willems-Vandijk K
   40. Oostra BA
   41. Willems SM
   42. Lamina C
   43. Winkler TW
   44. Psaty BM
   45. Tracy RP
   46. Brody J
   47. Chen I
   48. Viikari J
   49. Kähönen M
   50. Pramstaller PP
   51. Evans DM
   52. St Pourcain B
   53. Sattar N
   54. Wood AR
   55. Bandinelli S
   56. Carlson OD
   57. Egan JM
   58. Böhringer S
   59. van Heemst D
   60. Kedenko L
   61. Kristiansson K
   62. Nuotio ML
   63. Loo BM
   64. Harris T
   65. Garcia M
   66. Kanaya A
   67. Haun M
   68. Klopp N
   69. Wichmann HE
   70. Deloukas P
   71. Katsareli E
   72. Couper DJ
   73. Duncan BB
   74. Kloppenburg M
   75. Adair LS
   76. Borja JB
   77. Wilson JG
   78. Musani S
   79. Guo X
   80. Johnson T
   81. Semple R
   82. Teslovich TM
   83. Allison MA
   84. Redline S
   85. Buxbaum SG
   86. Mohlke KL
   87. Meulenbelt I
   88. Ballantyne CM
   89. Dedoussis GV
   90. Hu FB
   91. Liu Y
   92. Paulweber B
   93. Spector TD
   94. Slagboom PE
   95. Ferrucci L
   96. Jula A
   97. Perola M
   98. Raitakari O
   99. Florez JC
   100. Salomaa V
   101. Eriksson JG
   102. Frayling TM
   103. Hicks AA
   104. Lehtimäki T
   105. Smith GD
   106. Siscovick DS
   107. Kronenberg F
   108. van Duijn C
   109. Loos RJ
   110. Waterworth DM
   111. Meigs JB
   112. Dupuis J
   113. Richards JB
   114. Voight BF
   115. Scott LJ
   116. Steinthorsdottir V
   117. Dina C
   118. Welch RP
   119. Zeggini E
   120. Huth C
   121. Aulchenko YS
   122. Thorleifsson G
   123. McCulloch LJ
   124. Ferreira T
   125. Grallert H
   126. Amin N
   127. Wu G
   128. Willer CJ
   129. Raychaudhuri S
   130. McCarroll SA
   131. Hofmann OM
   132. Segrè AV
   133. van Hoek M
   134. Navarro P
   135. Ardlie K
   136. Balkau B
   137. Benediktsson R
   138. Bennett AJ
   139. Blagieva R
   140. Boerwinkle E
   141. Bonnycastle LL
   142. Boström KB
   143. Bravenboer B
   144. Bumpstead S
   145. Burtt NP
   146. Charpentier G
   147. Chines PS
   148. Cornelis M
   149. Crawford G
   150. Doney AS
   151. Elliott KS
   152. Elliott AL
   153. Erdos MR
   154. Fox CS
   155. Franklin CS
   156. Ganser M
   157. Gieger C
   158. Grarup N
   159. Green T
   160. Griffin S
   161. Groves CJ
   162. Guiducci C
   163. Hadjadj S
   164. Hassanali N
   165. Herder C
   166. Isomaa B
   167. Jackson AU
   168. Johnson PR
   169. Jørgensen T
   170. Kao WH
   171. Kong A
   172. Kraft P
   173. Kuusisto J
   174. Lauritzen T
   175. Li M
   176. Lieverse A
   177. Lindgren CM
   178. Lyssenko V
   179. Marre M
   180. Meitinger T
   181. Midthjell K
   182. Morken MA
   183. Narisu N
   184. Nilsson P
   185. Owen KR
   186. Payne F
   187. Petersen AK
   188. Platou C
   189. Proença C
   190. Prokopenko I
   191. Rathmann W
   192. Rayner NW
   193. Robertson NR
   194. Rocheleau G
   195. Roden M
   196. Sampson MJ
   197. Saxena R
   198. Shields BM
   199. Shrader P
   200. Sigurdsson G
   201. Sparsø T
   202. Strassburger K
   203. Stringham HM
   204. Sun Q
   205. Swift AJ
   206. Thorand B
   207. Tichet J
   208. Tuomi T
   209. van Dam RM
   210. van Haeften TW
   211. van Herpt T
   212. van Vliet-Ostaptchouk JV
   213. Walters GB
   214. Weedon MN
   215. Wijmenga C
   216. Witteman J
   217. Bergman RN
   218. Cauchi S
   219. Collins FS
   220. Gloyn AL
   221. Gyllensten U
   222. Hansen T
   223. Hide WA
   224. Hitman GA
   225. Hofman A
   226. Hunter DJ
   227. Hveem K
   228. Laakso M
   229. Morris AD
   230. Palmer CN
   231. Rudan I
   232. Sijbrands E
   233. Stein LD
   234. Tuomilehto J
   235. Uitterlinden A
   236. Walker M
   237. Watanabe RM
   238. Abecasis GR
   239. Boehm BO
   240. Campbell H
   241. Daly MJ
   242. Hattersley AT
   243. Pedersen O
   244. Barroso I
   245. Groop L
   246. Sladek R
   247. Thorsteinsdottir U
   248. Wilson JF
   249. Illig T
   250. Froguel P
   251. van Duijn CM
   252. Stefansson K
   253. Altshuler D
   254. Boehnke M
   255. McCarthy MI
   256. Soranzo N
   257. Wheeler E
   258. Glazer NL
   259. Bouatia-Naji N
   260. Mägi R
   261. Randall J
   262. Elliott P
   263. Rybin D
   264. Dehghan A
   265. Hottenga JJ
   266. Song K
   267. Goel A
   268. Lajunen T
   269. Doney A
   270. Cavalcanti-Proença C
   271. Kumari M
   272. Timpson NJ
   273. Zabena C
   274. Ingelsson E
   275. An P
   276. O'Connell J
   277. Luan J
   278. Elliott A
   279. McCarroll SA
   280. Roccasecca RM
   281. Pattou F
   282. Sethupathy P
   283. Ariyurek Y
   284. Barter P
   285. Beilby JP
   286. Ben-Shlomo Y
   287. Bergmann S
   288. Bochud M
   289. Bonnefond A
   290. Borch-Johnsen K
   291. Böttcher Y
   292. Brunner E
   293. Bumpstead SJ
   294. Chen YD
   295. Chines P
   296. Clarke R
   297. Coin LJ
   298. Cooper MN
   299. Crisponi L
   300. Day IN
   301. de Geus EJ
   302. Delplanque J
   303. Fedson AC
   304. Fischer-Rosinsky A
   305. Forouhi NG
   306. Franzosi MG
   307. Galan P
   308. Goodarzi MO
   309. Graessler J
   310. Grundy S
   311. Gwilliam R
   312. Hallmans G
   313. Hammond N
   314. Han X
   315. Hartikainen AL
   316. Hayward C
   317. Heath SC
   318. Hercberg S
   319. Hillman DR
   320. Hingorani AD
   321. Hui J
   322. Hung J
   323. Kaakinen M
   324. Kaprio J
   325. Kesaniemi YA
   326. Kivimaki M
   327. Knight B
   328. Koskinen S
   329. Kovacs P
   330. Kyvik KO
   331. Lathrop GM
   332. Lawlor DA
   333. Le Bacquer O
   334. Lecoeur C
   335. Li Y
   336. Mahley R
   337. Mangino M
   338. Martínez-Larrad MT
   339. McAteer JB
   340. McPherson R
   341. Meisinger C
   342. Melzer D
   343. Meyre D
   344. Mitchell BD
   345. Mukherjee S
   346. Naitza S
   347. Neville MJ
   348. Orrù M
   349. Pakyz R
   350. Paolisso G
   351. Pattaro C
   352. Pearson D
   353. Peden JF
   354. Pedersen NL
   355. Pfeiffer AF
   356. Pichler I
   357. Polasek O
   358. Posthuma D
   359. Potter SC
   360. Pouta A
   361. Province MA
   362. Rayner NW
   363. Rice K
   364. Ripatti S
   365. Rivadeneira F
   366. Rolandsson O
   367. Sandbaek A
   368. Sandhu M
   369. Sanna S
   370. Sayer AA
   371. Scheet P
   372. Seedorf U
   373. Sharp SJ
   374. Shields B
   375. Sigurðsson G
   376. Sijbrands EJ
   377. Silveira A
   378. Simpson L
   379. Singleton A
   380. Smith NL
   381. Sovio U
   382. Swift A
   383. Syddall H
   384. Syvänen AC
   385. Tönjes A
   386. Uitterlinden AG
   387. van Dijk KW
   388. Varma D
   389. Visvikis-Siest S
   390. Vitart V
   391. Vogelzangs N
   392. Waeber G
   393. Wagner PJ
   394. Walley A
   395. Ward KL
   396. Watkins H
   397. Wild SH
   398. Willemsen G
   399. Witteman JC
   400. Yarnell JW
   401. Zelenika D
   402. Zethelius B
   403. Zhai G
   404. Zhao JH
   405. Zillikens MC
   406. Borecki IB
   407. Meneton P
   408. Magnusson PK
   409. Nathan DM
   410. Williams GH
   411. Silander K
   412. Bornstein SR
   413. Schwarz P
   414. Spranger J
   415. Karpe F
   416. Shuldiner AR
   417. Cooper C
   418. Serrano-Ríos M
   419. Lind L
   420. Palmer LJ
   421. Hu FB
   422. Franks PW
   423. Ebrahim S
   424. Marmot M
   425. Kao WH
   426. Pramstaller PP
   427. Wright AF
   428. Stumvoll M
   429. Hamsten A
   430. Buchanan TA
   431. Valle TT
   432. Rotter JI
   433. Penninx BW
   434. Boomsma DI
   435. Cao A
   436. Scuteri A
   437. Schlessinger D
   438. Uda M
   439. Ruokonen A
   440. Jarvelin MR
   441. Peltonen L
   442. Mooser V
   443. Sladek R
   444. Musunuru K
   445. Smith AV
   446. Edmondson AC
   447. Stylianou IM
   448. Koseki M
   449. Pirruccello JP
   450. Chasman DI
   451. Johansen CT
   452. Fouchier SW
   453. Peloso GM
   454. Barbalic M
   455. Ricketts SL
   456. Bis JC
   457. Feitosa MF
   458. Orho-Melander M
   459. Melander O
   460. Li X
   461. Li M
   462. Cho YS
   463. Go MJ
   464. Kim YJ
   465. Lee JY
   466. Park T
   467. Kim K
   468. Sim X
   469. Ong RT
   470. Croteau-Chonka DC
   471. Lange LA
   472. Smith JD
   473. Ziegler A
   474. Zhang W
   475. Zee RY
   476. Whitfield JB
   477. Thompson JR
   478. Surakka I
   479. Spector TD
   480. Smit JH
   481. Sinisalo J
   482. Scott J
   483. Saharinen J
   484. Sabatti C
   485. Rose LM
   486. Roberts R
   487. Rieder M
   488. Parker AN
   489. Pare G
   490. O'Donnell CJ
   491. Nieminen MS
   492. Nickerson DA
   493. Montgomery GW
   494. McArdle W
   495. Masson D
   496. Martin NG
   497. Marroni F
   498. Lucas G
   499. Luben R
   500. Lokki ML
   501. Lettre G
   502. Launer LJ
   503. Lakatta EG
   504. Laaksonen R
   505. Kyvik KO
   506. König IR
   507. Khaw KT
   508. Kaplan LM
   509. Johansson Å
   510. Janssens AC
   511. Igl W
   512. Hovingh GK
   513. Hengstenberg C
   514. Havulinna AS
   515. Hastie ND
   516. Harris TB
   517. Haritunians T
   518. Hall AS
   519. Groop LC
   520. Gonzalez E
   521. Freimer NB
   522. Erdmann J
   523. Ejebe KG
   524. Döring A
   525. Dominiczak AF
   526. Demissie S
   527. Deloukas P
   528. de Faire U
   529. Crawford G
   530. Chen YD
   531. Caulfield MJ
   532. Boekholdt SM
   533. Assimes TL
   534. Quertermous T
   535. Seielstad M
   536. Wong TY
   537. Tai ES
   538. Feranil AB
   539. Kuzawa CW
   540. Taylor HA
   541. Gabriel SB
   542. Holm H
   543. Gudnason V
   544. Krauss RM
   545. Ordovas JM
   546. Munroe PB
   547. Kooner JS
   548. Tall AR
   549. Hegele RA
   550. Kastelein JJ
   551. Schadt EE
   552. Strachan DP
   553. Reilly MP
   554. Samani NJ
   555. Schunkert H
   556. Cupples LA
   557. Sandhu MS
   558. Ridker PM
   559. Rader DJ
   560. Kathiresan S
   561. DIAGRAM+ Consortium
   562. MAGIC Consortium
   563. GLGC Investigators
   564. MuTHER Consortium
   565. DIAGRAM Consortium
   566. GIANT Consortium
   567. Global B Pgen Consortium
   568. Procardis Consortium
   569. MAGIC investigators
   570. GLGC Consortium

   (2012) Novel loci for adiponectin levels and their influence on type 2 diabetes and metabolic traits: a multi-ethnic meta-analysis of 45,891 individuals

   PLOS Genetics 8:e1002607.

   https://doi.org/10.1371/journal.pgen.1002607

   • PubMed
   • Google Scholar
5. 1. Denzel MS
   2. Scimia MC
   3. Zumstein PM
   4. Walsh K
   5. Ruiz-Lozano P
   6. Ranscht B

   (2010) T-cadherin is critical for adiponectin-mediated cardioprotection in mice

   Journal of Clinical Investigation 120:4342–4352.

   https://doi.org/10.1172/JCI43464

   • PubMed
   • Google Scholar
6. 1. Fujii Y
   2. Kaneko M
   3. Neyazaki M
   4. Nogi T
   5. Kato Y
   6. Takagi J

   (2014) PA tag: a versatile protein tagging system using a super high affinity antibody against a dodecapeptide derived from human podoplanin

   Protein Expression and Purification 95:240–247.

   https://doi.org/10.1016/j.pep.2014.01.009

   • PubMed
   • Google Scholar
7. 1. Fujishima Y
   2. Maeda N
   3. Matsuda K
   4. Masuda S
   5. Mori T
   6. Fukuda S
   7. Sekimoto R
   8. Yamaoka M
   9. Obata Y
   10. Kita S
   11. Nishizawa H
   12. Funahashi T
   13. Ranscht B
   14. Shimomura I

   (2017) Adiponectin association with T-cadherin protects against neointima proliferation and atherosclerosis

   The FASEB Journal 31:1571–1583.

   https://doi.org/10.1096/fj.201601064R

   • PubMed
   • Google Scholar
8. 1. Fukuda S
   2. Kita S
   3. Obata Y
   4. Fujishima Y
   5. Nagao H
   6. Masuda S
   7. Tanaka Y
   8. Nishizawa H
   9. Funahashi T
   10. Takagi J
   11. Maeda N
   12. Shimomura I

   (2017) The unique prodomain of T-cadherin plays a key role in adiponectin binding with the essential extracellular cadherin repeats 1 and 2

   Journal of Biological Chemistry 292:7840–7849.

   https://doi.org/10.1074/jbc.M117.780734

   • PubMed
   • Google Scholar
9. 1. Gardai SJ
   2. McPhillips KA
   3. Frasch SC
   4. Janssen WJ
   5. Starefeldt A
   6. Murphy-Ullrich JE
   7. Bratton DL
   8. Oldenborg PA
   9. Michalak M
   10. Henson PM

   (2005) Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte

   Cell 123:321–334.

   https://doi.org/10.1016/j.cell.2005.08.032

   • PubMed
   • Google Scholar
10. 1. Hu E
    2. Liang P
    3. Spiegelman BM

    (1996) AdipoQ is a novel adipose-specific gene dysregulated in obesity

    Journal of Biological Chemistry 271:10697–10703.

    https://doi.org/10.1074/jbc.271.18.10697

    • PubMed
    • Google Scholar
11. 1. Hug C
    2. Wang J
    3. Ahmad NS
    4. Bogan JS
    5. Tsao TS
    6. Lodish HF

    (2004) T-cadherin is a receptor for hexameric and high-molecular-weight forms of Acrp30/adiponectin

    PNAS 101:10308–10313.

    https://doi.org/10.1073/pnas.0403382101

    • PubMed
    • Google Scholar
12. 1. Hulpiau P
    2. van Roy F

    (2009) Molecular evolution of the cadherin superfamily

    The International Journal of Biochemistry & Cell Biology 41:349–369.

    https://doi.org/10.1016/j.biocel.2008.09.027

    • PubMed
    • Google Scholar
13. 1. Kita S
    2. Maeda N
    3. Shimomura I

    (2019) Interorgan communication by exosomes, adipose tissue, and adiponectin in metabolic syndrome

    Journal of Clinical Investigation 129:4041–4049.

    https://doi.org/10.1172/JCI129193

    • PubMed
    • Google Scholar
14. 1. Kitamoto A
    2. Kitamoto T
    3. Nakamura T
    4. Matsuo T
    5. Nakata Y
    6. Hyogo H
    7. Ochi H
    8. Kamohara S
    9. Miyatake N
    10. Kotani K
    11. Mineo I
    12. Wada J
    13. Ogawa Y
    14. Yoneda M
    15. Nakajima A
    16. Funahashi T
    17. Miyazaki S
    18. Tokunaga K
    19. Masuzaki H
    20. Ueno T
    21. Chayama K
    22. Hamaguchi K
    23. Yamada K
    24. Hanafusa T
    25. Oikawa S
    26. Sakata T
    27. Tanaka K
    28. Matsuzawa Y
    29. Hotta K

    (2016) CDH13 polymorphisms are associated with adiponectin levels and metabolic syndrome traits independently of visceral fat mass

    Journal of Atherosclerosis and Thrombosis 23:309–319.

    https://doi.org/10.5551/jat.31567

    • PubMed
    • Google Scholar
15. 1. Maeda K
    2. Okubo K
    3. Shimomura I
    4. Funahashi T
    5. Matsuzawa Y
    6. Matsubara K

    (1996) cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1 (AdiPose most abundant gene transcript 1)

    Biochemical and Biophysical Research Communications 221:286–289.

    https://doi.org/10.1006/bbrc.1996.0587

    • PubMed
    • Google Scholar
16. 1. Matsuda K
    2. Fujishima Y
    3. Maeda N
    4. Mori T
    5. Hirata A
    6. Sekimoto R
    7. Tsushima Y
    8. Masuda S
    9. Yamaoka M
    10. Inoue K
    11. Nishizawa H
    12. Kita S
    13. Ranscht B
    14. Funahashi T
    15. Shimomura I

    (2015) Positive feedback regulation between adiponectin and T-cadherin impacts adiponectin levels in tissue and plasma of male mice

    Endocrinology 156:934–946.

    https://doi.org/10.1210/en.2014-1618

    • PubMed
    • Google Scholar
17. 1. Mendlov F
    2. Conconi M

    (2010)

    Calreticulin: a multifaceted protein

    Nature Education 4:1.

    • Google Scholar
18. 1. Michalak M
    2. Groenendyk J
    3. Szabo E
    4. Gold LI
    5. Opas M

    (2009) Calreticulin, a multi-process calcium-buffering chaperone of the endoplasmic reticulum

    Biochemical Journal 417:651–666.

    https://doi.org/10.1042/BJ20081847

    • PubMed
    • Google Scholar
19. 1. Morisaki H
    2. Yamanaka I
    3. Iwai N
    4. Miyamoto Y
    5. Kokubo Y
    6. Okamura T
    7. Okayama A
    8. Morisaki T

    (2012) CDH13 gene coding T-cadherin influences variations in plasma adiponectin levels in the japanese population

    Human Mutation 33:402–410.

    https://doi.org/10.1002/humu.21652

    • PubMed
    • Google Scholar
20. 1. Nakano Y
    2. Tobe T
    3. Choi-Miura NH
    4. Mazda T
    5. Tomita M

    (1996) Isolation and characterization of GBP28, a novel gelatin-binding protein purified from human plasma

    Journal of Biochemistry 120:803–812.

    https://doi.org/10.1093/oxfordjournals.jbchem.a021483

    • PubMed
    • Google Scholar
21. 1. Obata Y
    2. Kita S
    3. Koyama Y
    4. Fukuda S
    5. Takeda H
    6. Takahashi M
    7. Fujishima Y
    8. Nagao H
    9. Masuda S
    10. Tanaka Y
    11. Nakamura Y
    12. Nishizawa H
    13. Funahashi T
    14. Ranscht B
    15. Izumi Y
    16. Bamba T
    17. Fukusaki E
    18. Hanayama R
    19. Shimada S
    20. Maeda N
    21. Shimomura I

    (2018) Adiponectin/T-cadherin system enhances exosome biogenesis and decreases cellular ceramides by exosomal release

    JCI Insight 3:.

    https://doi.org/10.1172/jci.insight.99680

    • PubMed
    • Google Scholar
22. 1. Parker-Duffen JL
    2. Nakamura K
    3. Silver M
    4. Kikuchi R
    5. Tigges U
    6. Yoshida S
    7. Denzel MS
    8. Ranscht B
    9. Walsh K

    (2013) T-cadherin is essential for adiponectin-mediated revascularization

    Journal of Biological Chemistry 288:24886–24897.

    https://doi.org/10.1074/jbc.M113.454835

    • PubMed
    • Google Scholar
23. 1. Scherer PE
    2. Williams S
    3. Fogliano M
    4. Baldini G
    5. Lodish HF

    (1995) A novel serum protein similar to C1q, produced exclusively in adipocytes

    Journal of Biological Chemistry 270:26746–26749.

    https://doi.org/10.1074/jbc.270.45.26746

    • PubMed
    • Google Scholar
24. 1. Straub LG
    2. Scherer PE

    (2019) Metabolic messengers: adiponectin

    Nature Metabolism 1:334–339.

    https://doi.org/10.1038/s42255-019-0041-z

    • Google Scholar
25. 1. Suzuki S
    2. Wilson-Kubalek EM
    3. Wert D
    4. Tsao TS
    5. Lee DH

    (2007) The oligomeric structure of high molecular weight adiponectin

    FEBS Letters 581:809–814.

    https://doi.org/10.1016/j.febslet.2007.01.046

    • PubMed
    • Google Scholar
26. 1. Takemura Y
    2. Ouchi N
    3. Shibata R
    4. Aprahamian T
    5. Kirber MT
    6. Summer RS
    7. Kihara S
    8. Walsh K

    (2007) Adiponectin modulates inflammatory reactions via calreticulin receptor-dependent clearance of early apoptotic bodies

    Journal of Clinical Investigation 117:375–386.

    https://doi.org/10.1172/JCI29709

    • PubMed
    • Google Scholar
27. 1. Tanaka Y
    2. Kita S
    3. Nishizawa H
    4. Fukuda S
    5. Fujishima Y
    6. Obata Y
    7. Nagao H
    8. Masuda S
    9. Nakamura Y
    10. Shimizu Y
    11. Mineo R
    12. Natsukawa T
    13. Funahashi T
    14. Ranscht B
    15. Fukada SI
    16. Maeda N
    17. Shimomura I

    (2019) Adiponectin promotes muscle regeneration through binding to T-cadherin

    Scientific Reports 9:16.

    https://doi.org/10.1038/s41598-018-37115-3

    • PubMed
    • Google Scholar
28. 1. Tanford C
    2. Reynolds JA

    (1976) Characterization of membrane proteins in detergent solutions

    Biochimica Et Biophysica Acta (BBA) - Reviews on Biomembranes 457:133–170.

    https://doi.org/10.1016/0304-4157(76)90009-5

    • Google Scholar
29. 1. Tang YT
    2. Hu T
    3. Arterburn M
    4. Boyle B
    5. Bright JM
    6. Emtage PC
    7. Funk WD

    (2005) PAQR proteins: a novel membrane receptor family defined by an ancient 7-transmembrane pass motif

    Journal of Molecular Evolution 61:372–380.

    https://doi.org/10.1007/s00239-004-0375-2

    • PubMed
    • Google Scholar
30. 1. van Niel G
    2. D'Angelo G
    3. Raposo G

    (2018) Shedding light on the cell biology of extracellular vesicles

    Nature Reviews Molecular Cell Biology 19:213–228.

    https://doi.org/10.1038/nrm.2017.125

    • PubMed
    • Google Scholar
31. 1. Yamauchi T
    2. Kamon J
    3. Ito Y
    4. Tsuchida A
    5. Yokomizo T
    6. Kita S
    7. Sugiyama T
    8. Miyagishi M
    9. Hara K
    10. Tsunoda M
    11. Murakami K
    12. Ohteki T
    13. Uchida S
    14. Takekawa S
    15. Waki H
    16. Tsuno NH
    17. Shibata Y
    18. Terauchi Y
    19. Froguel P
    20. Tobe K
    21. Koyasu S
    22. Taira K
    23. Kitamura T
    24. Shimizu T
    25. Nagai R
    26. Kadowaki T

    (2003) Cloning of adiponectin receptors that mediate antidiabetic metabolic effects

    Nature 423:762–769.

    https://doi.org/10.1038/nature01705

    • PubMed
    • Google Scholar
32. 1. Yamauchi T
    2. Nio Y
    3. Maki T
    4. Kobayashi M
    5. Takazawa T
    6. Iwabu M
    7. Okada-Iwabu M
    8. Kawamoto S
    9. Kubota N
    10. Kubota T
    11. Ito Y
    12. Kamon J
    13. Tsuchida A
    14. Kumagai K
    15. Kozono H
    16. Hada Y
    17. Ogata H
    18. Tokuyama K
    19. Tsunoda M
    20. Ide T
    21. Murakami K
    22. Awazawa M
    23. Takamoto I
    24. Froguel P
    25. Hara K
    26. Tobe K
    27. Nagai R
    28. Ueki K
    29. Kadowaki T

    (2007) Targeted disruption of AdipoR1 and AdipoR2 causes abrogation of adiponectin binding and metabolic actions

    Nature Medicine 13:332–339.

    https://doi.org/10.1038/nm1557

    • PubMed
    • Google Scholar
33. 1. Yamauchi T
    2. Iwabu M
    3. Okada-Iwabu M
    4. Kadowaki T

    (2014) Adiponectin receptors: a review of their structure, function and how they work

    Best Practice & Research Clinical Endocrinology & Metabolism 28:15–23.

    https://doi.org/10.1016/j.beem.2013.09.003

    • PubMed
    • Google Scholar

[Editors’ note: this article was originally rejected after discussions between the reviewers, but the authors were invited to resubmit after an appeal against the decision.]

Thank you for submitting your work entitled "Native adiponectin existing in serum binds with T-cadherin, but not with AdipoRs nor Calreticulin" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Morris Birnbaum (Reviewer #3).

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

This study focused on elucidating the physiological binding partner(s) for native adiponectin. With cell culture based studies, they have revealed that serum adiponectin exclusively bound to T-cadherin, but not to the "classical" AdipoR receptors nor Calreticulin. The strength of the study is that they have shown binding of "native" adiponectin to T-cadherin as this has not been shown before. However, the study has several weaknesses which preclude publication in eLife. First, the binding system used is somewhat "dirty" limiting careful biochemical analyses. Second, the overall advance of the study is unclear as it has already been shown that adiponectin binds to T-cadherin. Third, all reviewers highlighted weaknesses with the binding assay pinpointing the inability of the authors to reach saturation binding, an essential criterion for a receptor-ligand interaction. Finally, as highlighted by reviewer 1, much more work is required to prove a lack of interaction between adiponectin and the other receptor systems.

Reviewer #1:

This manuscript presents data showing serum adiponectin binds to T-cadherin overexpressed in HEK293 cells as well as endogenous T-cadherin in C2C12 cells. These findings are solid and convincing since in such experiments adiponectin is in native forms and being adsorbed directly in such forms. The claim that T-cadherin is a "receptor" for adiponectin is justified by these data and conforms to other solid papers in the field. These data do not add much to the literature as it is already established that adiponectin binds to T-cadherin, although this is a new way of demonstrating the phenomenon. But the claim is convincing.

On the other hand, the failure to show binding of serum adiponectin to other purported receptor molecules (R1 and R2 as well as Calreticulin) is more difficult to interpret. The claim that these are not true receptors could be true, but it is impossible to prove a negative. What if there are "accessory" proteins required to confer binding activity to these other putative receptors? Expression of the receptors alone would not be sufficient to display binding in that case. Normally, ligand-receptor interactions are quantified by binding of pure ligand with pure receptor, which allows binding constants to be calculated. This is not the case here, since the ligand is in a complex mixture (serum) and the "receptor" is also within a complex structure (cell membrane). These complications raise concerns about the conclusion, and have caused other groups to use gene KO mice to interrogate putative "receptor" function.

On balance, this paper should be published since it raises an important question for the field. Publication would also highlight the need to determine binding affinities for the putative receptors. However, the study doesn't add mechanistic information to the field, and doesn't define why dysfunction of adiponectin occurs when the R1/R2 receptors are deleted in mice. For those reasons, it could be argued that this paper belongs in a specialty journal.

One other point is that careful editing of this manuscript for proper English usage would be mandatory prior to publication.

Reviewer #2:

In this study, Kita et al. have focused on the elucidation of physiological binding partner(s) for native adiponectin. With cell culture based studies, they have revealed that serum adiponectin exclusively bound to T-cadherin, but not to AdipoR nor Calreticulin. While this study contains potential interests, several issues need to be properly addressed to enhance the significance of this study.

1) Due to molecular structure of T-cadherin protein, it remains elusive to guess the mode(s) of action for adiponectin signaling. More discussion and/or speculation would be more helpful for general readers to understand adiponectin action and signaling.

2) Along the same line, it is unclear whether T-cadherin might behave as a typical receptor protein for adiponectin. For example, typical receptors are saturated by ligands. However, provided data (Figures 1, 2 and 3) did not show any saturation pattern nor dose response. This issue needs to be properly addressed by experimental data.

3) The authors demonstrated that native adiponectin bound to T-cadherin. Have you compared the binding affinity with native and recombinant adiponectin proteins to three potential receptors? If there is any difference, it needs to be described and discussed.

Reviewer #3:

In spite of the identification of adiponectin quite a number of years ago, the nature of its receptors remains controversial. The rationale for the current study is that the major criterion for evaluating a potential receptor should be that it binds native adiponectin and not just recombinant product. This seems quite reasonable and therefore the strength of the paper is that it seeks to accomplish that. The weakness is that the nature of the protocol involves performing the binding study on a very complex, on might say "dirty" system, in which it is challenging to infer the actual biochemical even being measured. At a minimum, the authors' need to be much more precise in their language and judicious in what they claim the data show. For example, the authors state "These results clearly indicate that T-cadherin can bind native adiponectin." This is a gross overinterpretation of this experiment. What the authors show is that overexpression of T-cadherin in cells allows them to bind increased amounts of adiponectin. There is no information to indicated that the adiponectin is binding directly to T-cadherin or, for that matter, that T-cadherin is involved in the binding of hormone. This interpretation might be plausible, but it is far from proven.

The authors do demonstrate that the binding is concentration dependent, which is important for true receptor binding, but they do not establish saturability, which is essential to arguing the physiological relevance of the event being measured. Thus, it would add to the weight of the argument of they could show a full binding curve up to the point of saturability. It is relatively straightforward to vary expression to make sure the receptor level is low enough that it can be saturated with the adiponectin levels present in serum.

[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]

Congratulations, we are pleased to inform you that your article, "Native adiponectin in serum binds to cells expressing T-cadherin, but not AdipoRs or calreticulin", has been accepted for publication in eLife as a Short Report article.

While the reviewers still identified some shortcomings with your study, it was generally considered that your paper makes an important contribution to the field. In particular, there is an abundance of evidence in the literature to show that adiponectin signals via the ADIPOR1 and 2 receptors. Your work questions the breadth of this conclusion and shows that as a minimum the field needs to carefully consider the role of this alternate adiponectin receptor, T cadherin. This paper clearly shows that adiponectin binds to T-cadherin with considerable affinity under somewhat physiological conditions. As to whether the origin or nature of the ligand influences the binding specificity is unclear but this is certainly something important to consider that this work highlights. While, as pointed out by two of the reviewers, there is a long way to go to ascertain how the adiponectin-T-cadherin interaction signals in cells this is perhaps something for a future study.

Please address as best you can each of the points raised by the reviewers as noted below. Also I noted that some restructuring of your figures is necessary to consolidate space and to improve the presentation of the data. Furthermore, I still believe you could shorten the article without loss of clarity.

Reviewer #1:

This paper provides data supporting the view that binding of adiponectin to cells is through T-cadherin rather than other purported receptor types. There are three issues that diminish the impact of the paper:

1) The first paragraph is an overly long "stream of consciousness" type of Introduction that should be heavily edited and organized into multiple paragraphs around the several discrete ideas.

2) The major impact of this study would be that bio-effects of adiponectin are mediated through T-cadherin, not the other putative receptors, but unfortunately no bio-effects are studied in these experiments. While the results are very interesting and certainly provocative, a definitive experiment on this key point showing that T-cadherin but not the other receptors mediates important bio-effects is missing.

3) The cell biology/microscopy data are too limited. Multiple fields should be shown and appropriate controls with null signal. The data should be quantified from many fields as well.

Reviewer #2:

In this work, Kita et al. have demonstrated that native adiponectin would exclusively bind to T-cadherin. With cell culture model, they showed that serum or purified adiponectin preferentially bound to T-cadherin but not to AdipoR nor Calreticulin. Although they provided more data and tried to strengthen Discussion part in the resubmitted manuscript, it still has technical limitations to reach the firm conclusion.

1) Although it has been reported that adiponectin binds to AdipoRs, calreticulin, and T-cadherin, they argued that the major binding protein for native adiponectin is T-cadherin. If the binding affinity of native adiponectin to adipoRs or calreticulin would be much weaker than T-cadherin, it seems that the experimental condition in this study might be too harsh to detect weak interaction with others. They have to adopt alternative methods to affirm the extent of protein interactions. Along the same line, they need to measure/compare binding affinity of recombinant adiponectin with other proteins including AdipoR and Calreticulin.

2) To convince that native adiponectin exclusively binds to T-cadherin, it is crucial to provide positive control data.

3) It has been suggested to examine intracellular signaling cascades such as phospho-AMPK and phospho-p38 upon the interaction between native adiponectin and potential receptors.

Reviewer #3:

I was ambivalent about this manuscript the first time around and the authors have only improved it with the additional experiments. In all honesty, this will never be the cleanest study and will not have the highest level of impact due to lack of mechanism, whatever the authors do to address the issues. However, I continue to believe that it brings up a valid point about the ambiguity of the true adiponectin receptor and the data are strong enough to provide a valid challenge to the dogma. On balance, I recommend acceptance.

[Editors’ note: the author responses to the first round of peer review follow.]

This study focused on elucidating the physiological binding partner(s) for native adiponectin. With cell culture based studies, they have revealed that serum adiponectin exclusively bound to T-cadherin, but not to the "classical" AdipoR receptors nor Calreticulin. The strength of the study is that they have shown binding of "native" adiponectin to T-cadherin as this has not been shown before.

However, the study has several weaknesses which preclude publication in eLife. First, the binding system used is somewhat "dirty" limiting careful biochemical analyses.

We used HMW adiponectin purified from serum (Fukuda et al., 2017, PMID: 28325833) and full-length recombinant adiponectin produced by HEK293 cells in our revised manuscript, and they gave essentially the same results. Furthermore, we would like to emphasize that the ligand in serum is the most natural ligand that maintains its ability to interact with the physiological binding partner. Numerous constituents, including a large number of proteins, exert adequate blocking effects to investigate whether the interaction between the physiological ligand and binding protein is specific. Moreover, the use of a recombinant ligand may make the experiment unreproducible because the same recombinant ligand cannot be made at another site. The ligand-receptor interaction in a more purified system can have mean if the ligand can bind with the receptor in a more physiological situation like in serum as our experiment. The results of our new experiment indicate that recombinant adiponectin has a limited affinity to T-cadherin due to the presence of less of the HMW form.

The results obtained were shown in Figure 1B and 1D. We added the following sentences.

Results and Discussion

“We directly examined mouse serum as the ligand solution, including the most native adiponectin, purified adiponectin from mouse serum (Fukuda et al., 2017), and full-length recombinant adiponectin produced in HEK293 cells. […] Recombinant adiponectin contained a lower amount of HMW multimer adiponectin than mouse serum and purified adiponectin from serum (Figure 1B).

Results and Discussion

“The treatment of cells with different preparations of adiponectin at 4°C for 1 hr resulted in the binding of prepared adiponectin only to cells expressing mouse T-cadherin (Figure 1D). Mouse serum and purified adiponectin showed similar binding, whereas recombinant adiponectin containing a lower amount of 6-mer and the HMW multimer exhibited markedly weaker binding (Figure 1D).”

Second, the overall advance of the study is unclear as it has already been shown that adiponectin binds to T-cadherin.

The binding of HMW recombinant adiponectin to T-cadherin was initially discovered in 2004 (Hug C et al., 2004); however, it has not been investigated in detail because it lacks an intracellular domain. We previously reported a molecular interaction between serum derived purified HMW adiponectin and Fc-fusion purified T-cadherin with high affinity (KD=1.0 nM) (Fukuda et al., 2017). Moreover, we demonstrated that T-cadherin mediated the exosome-stimulating function of adiponectin (Obata et al., 2018). However, as discussed in the Abstract, reported receptors for adiponectin have not yet been simultaneously examined on their adiponectin binding under equal conditions. The present study established by WB that native HMW (> 6mer) adiponectin in serum and highly purified serum-derived multimer adiponectin bound to cells expressing T-cadherin, but not to those expressing AdipoRs or calreticulin. An important issue in the present study is that native HMW adiponectin, either in serum or highly purified, did not appear to bind to cells expressing AdipoRs or calreticulin, but did bind to those expressing T-cadherin in the same set of experiments. Based on these results, we speculate that AdipoRs and/or calreticulin may function as adiponectin receptors for LMW forms such as globular adiponectin, possibly on the site of cells after HMW adiponectin binding to cell-surface T-cadherin is cleaved. We discussed this possibility in our response to comment 4 by the Editor.

Third, all reviewers highlighted weaknesses with the binding assay pinpointing the inability of the authors to reach saturation binding, an essential criterion for a receptor-ligand interaction.

We showed the saturable binding of native adiponectin in serum in Figure 1E. The binding isotherm obtained is consistent with the high-affinity interaction we reported previously using purified HMW adiponectin with purified T-cadherin (Fukuda et al., 2017).

The results obtained were shown in Figure 1E. We added the following sentence.

Results and Discussion

“The dose-response study revealed the specific and saturable binding of native adiponectin in serum to cells expressing T-cadherin (Figure 1E).”

Finally, as highlighted by reviewer 1, much more work is required to prove a lack of interaction between adiponectin and the other receptor systems.

We simultaneously compared the candidate receptors. The expression of T-cadherin gave a strong band of bound adiponectin, which is consistent with our previous finding showing that recombinant T-cadherin binds HMW adiponectin in a 1: 1 ratio with high affinity. If the receptor has meaningful affinity, it will bind and give a visible band on WB, similar to that of T-cadherin. However, there was no detectable band on the AdipoR or calreticulin lane. Furthermore, AdipoR was discovered by expression cloning. Therefore, the ectopic introduction of AdipoRs promotes adiponectin binding. The Nature study also showed that HEK293 cells overexpressing AdipoRs bound E. coli recombinant globular adiponectin (Supplementary Figure 2A and 2B, Yamauchi et al., 2003). Therefore, difficulties are associated with speculating about the requirement for “accessory” proteins to confer adiponectin binding activity to AdipoRs.

Numerous studies have indicated that AdipoRs mediate adiponectin signaling. Therefore, there may be additional activating mechanisms of AdipoRs by adiponectin, i.e., reductive or proteolytic generation of the trimer, monomer, and/or globular adiponectin. The present study focused on the direct binding of native HMW adiponectin (more than a hexamer) and the results obtained suggest the activation of AdipoRs by these LMW forms, possibly on the site of cells after HMW adiponectin binding to cell surface T-cadherin is cleaved.

On the other hand, in the case of calreticulin, this was evidenced by a neutralizing antibody treatment inhibiting the adiponectin interaction with cells (Takemura et al., 2007). Therefore, “accessory” proteins may be required to confer adiponectin binding activity to calreticulin. We also revised our discussion on this point to keep the possibility of calreticulin mediating the adiponectin interaction with cells.

We added a detailed discussion on this important assumption as follows.

Results and Discussion

“Adiponectin is an atypically abundant circulating factor that is exclusively secreted from adipocytes as a trimer, hexamer, and HMW multimer. […] Therefore, some “accessory” proteins may be required to confer adiponectin binding activity to calreticulin.”

We also revised the Abstract as follows.

“Adiponectin is an adipocyte-derived atypically abundant circulating factor that protects various organs and tissues through its receptors, AdipoRs, calreticulin, and T-cadherin. […] Collectively, these results suggest that T-cadherin is the major binding partner of native adiponectin in serum.”

We also revised conclusive sentences at the bottom of the Results and Discussion sections as follows.

“The present results may further contribute to clarifying the activating mechanism of AdipoRs by LMW adiponectin, generated by reduction or proteolytic cleavage, followed by HMW adiponectin binding to cell surface T-cadherin.”

Reviewer #1:

This manuscript presents data showing serum adiponectin binds to T-cadherin overexpressed in HEK293 cells as well as endogenous T-cadherin in C2C12 cells. These findings are solid and convincing since in such experiments adiponectin is in native forms and being adsorbed directly in such forms. The claim that T-cadherin is a "receptor" for adiponectin is justified by these data and conforms to other solid papers in the field. These data do not add much to the literature as it is already established that adiponectin binds to T-cadherin, although this is a new way of demonstrating the phenomenon. But the claim is convincing.

The binding of HMW recombinant adiponectin to T-cadherin was initially discovered in 2004 (Hug C et al., 2004); however, it has not been investigated in detail because it lacks an intracellular domain. We previously reported a molecular interaction between serum-derived purified HMW adiponectin and Fc-fusion purified T-cadherin with high affinity (KD=1.0 nM) (Fukuda et al., 2017). Moreover, we demonstrated that T-cadherin mediated the exosome-stimulating function of adiponectin (Obata et al., 2018). However, as discussed in the Abstract, reported receptors for adiponectin have not yet been simultaneously examined under equal conditions on their adiponectin binding. The present study established by WB that native HMW (> 6mer) adiponectin in serum and highly purified serum derived multimer adiponectin bound to cells expressing T-cadherin, but not to those expressing AdipoRs or calreticulin. An important issue in the present study is that native HMW adiponectin, either in serum or highly purified, did not appear to bind to cells expressing AdipoRs or calreticulin, but did bind to those expressing T-cadherin in the same set of experiments. Based on these results, we speculate that AdipoRs and/or calreticulin may function as adiponectin receptors for LMW forms such as globular adiponectin, possibly on the site of cells after HMW adiponectin binding to cell surface T-cadherin is cleaved. We discussed this possibility in our response to reviewer #1’s comment 3.

On the other hand, the failure to show binding of serum adiponectin to other purported receptor molecules (R1 and R2 as well as Calreticulin) is more difficult to interpret. The claim that these are not true receptors could be true, but it is impossible to prove a negative. What if there are "accessory" proteins required to confer binding activity to these other putative receptors? Expression of the receptors alone would not be sufficient to display binding in that case. Normally, ligand-receptor interactions are quantified by binding of pure ligand with pure receptor, which allows binding constants to be calculated. This is not the case here, since the ligand is in a complex mixture (serum) and the "receptor" is also within a complex structure (cell membrane). These complications raise concerns about the conclusion, and have caused other groups to use gene KO mice to interrogate putative "receptor" function.

We appreciate the valuable comment. In the revised manuscript, we compared the binding of native adiponectin in serum, purified native adiponectin, and recombinant full-length adiponectin produced in HEK293 cells. The results obtained indicated that only cells expressing T-cadherin and not the other receptors bound these preparations of adiponectin. However, recombinant adiponectin with a lower amount of HMW multimer adiponectin showed weak binding at a physiological concentration (20 µg/mL) of adiponectin. The results obtained were shown in Figure 1B and 1D. We added the following sentences.

Results and Discussion

“We directly examined mouse serum as the ligand solution, including the most native adiponectin, purified adiponectin from mouse serum (Fukuda et al., 2017), and full-length recombinant adiponectin produced in HEK293 cells. […] Recombinant adiponectin contained a lower amount of HMW multimer adiponectin than mouse serum and purified adiponectin from serum (Figure 1B).”

Results and Discussion

“The treatment of cells with different preparations of adiponectin at 4°C for 1 hr resulted in the binding of prepared adiponectin only to cells expressing mouse T-cadherin (Figure 1D). Mouse serum and purified adiponectin showed similar binding, whereas recombinant adiponectin containing a lower amount of 6-mer and the HMW multimer exhibited markedly weaker binding (Figure 1D).”

Furthermore, we would like to emphasize that the ligand in serum is the most natural ligand that maintains its ability to interact with the physiological binding partner. Numerous constituents, including a large number of proteins, exert adequate blocking effects to investigate whether the interaction between the physiological ligand and binding protein is specific. Moreover, the use of a recombinant ligand may make the experiment unreproducible because the same recombinant ligand cannot be made at another site. The ligand-receptor interaction in a more purified system can have mean if the ligand can bind with the receptor in a more physiological situation like in serum as our experiment The results of our new experiment indicate that recombinant adiponectin has a limited affinity to T-cadherin due to the presence of less of the HMW form.

The expression of T-cadherin gave a strong band of bound adiponectin, which is consistent with our previous finding showing that recombinant T-cadherin binds HMW adiponectin in a 1: 1 ratio with high affinity. If the receptor has meaningful affinity, it will bind and give a visible band on WB, similar to that of T-cadherin. However, there was no detectable band on the AdipoR or calreticulin lane. Furthermore, AdipoR was discovered by expression cloning. Therefore, the ectopic introduction of AdipoRs promotes adiponectin binding. The Nature study also showed that HEK293 cells overexpressing AdipoRs bound E. coli recombinant globular adiponectin (Supplementary Figure 2A and 2B, Yamauchi et al., 2003). Therefore, difficulties are associated with speculating about the requirement for “accessory” proteins to confer adiponectin binding activity to AdipoRs.

On the other hand, in the case of calreticulin, this was evidenced by a neutralizing antibody treatment inhibiting the adiponectin interaction with cells (Takemura Y et al., 2007). Therefore, “accessory” proteins may be required to confer adiponectin binding activity to calreticulin. We also revised our discussion on this point to keep the possibility of calreticulin mediating the adiponectin interaction with cells.

We added a detailed discussion on this important assumption as follows.

Results and Discussion

“We simultaneously compared three adiponectin receptors. The expression of T-cadherin gave a strong band of bound adiponectin, which is consistent with our previous finding showing that recombinant Tcadherin binds HMW adiponectin in a 1: 1 ratio with high affinity (Fukuda et al., 2017). […] Therefore, difficulties are associated with speculating about the requirement for some “accessory” proteins to confer adiponectin binding activity to AdipoRs.”

Results and Discussion

“On the other hand, in the case of calreticulin, this was evidenced by a neutralizing antibody treatment inhibiting the adiponectin interaction with cells (Takemura et al., 2007). Therefore, some “accessory” proteins may be required to confer adiponectin binding activity to calreticulin.”

On balance, this paper should be published since it raises an important question for the field. Publication would also highlight the need to determine binding affinities for the putative receptors. However, the study doesn't add mechanistic information to the field, and doesn't define why dysfunction of adiponectin occurs when the R1/R2 receptors are deleted in mice. For those reasons, it could be argued that this paper belongs in a specialty journal.

We appreciate the valuable comment. Numerous studies indicated that AdipoRs mediate adiponectin signaling. Therefore, there may be additional activating mechanisms of AdipoRs by adiponectin, i.e., reductive or proteolytic generation of the trimer, monomer, and/or globular adiponectin. The present study focused on the direct binding of native HMW adiponectin (more than hexamer) and the results obtained suggest the activation of AdipoRs by these LMW forms, possibly on the site of cells after HMW adiponectin binding to cell-surface T-cadherin is cleaved. We added a detailed discussion on this important assumption as follows.

Results and Discussion

“Since numerous studies indicated that AdipoRs mediate adiponectin signaling in a number of cell types, an additional activating mechanism for AdipoRs by adiponectin, i.e., the reductive or proteolytic generation of the trimer, monomer, and/or globular adiponectin, may exist. The results of the present study, which focused on the direct binding of native HMW adiponectin, may indicate the activation of AdipoRs by low-molecular-weight (LMW) forms.”

We also revised the Abstract as follows.

“Adiponectin is an adipocyte-derived atypically abundant circulating factor that protects various organs and tissues through its receptors, AdipoRs, calreticulin, and T-cadherin. […] Collectively, these results suggest that T-cadherin is the major binding partner of native adiponectin in serum.”

We also revised conclusive sentences at the bottom of the Results and Discussion sections as follows.

“The present results may further contribute to clarifying the activating mechanism of AdipoRs by LMW adiponectin, generated by reduction or proteolytic cleavage, followed by HMW adiponectin binding to cell surface T-cadherin.”

One other point is that careful editing of this manuscript for proper English usage would be mandatory prior to publication.

The revised manuscript was proofread by a native speaker of English.

Reviewer #2:

In this study, Kita et al. have focused on the elucidation of physiological binding partner(s) for native adiponectin. With cell culture based studies, they have revealed that serum adiponectin exclusively bound to T-cadherin, but not to AdipoR nor Calreticulin. While this study contains potential interests, several issues need to be properly addressed to enhance the significance of this study.

1) Due to molecular structure of T-cadherin protein, it remains elusive to guess the mode(s) of action for adiponectin signaling. More discussion and/or speculation would be more helpful for general readers to understand adiponectin action and signaling.

We appreciate the valuable comment. We added a detailed discussion on the mode of action of adiponectin through T-cadherin as below.

Results and Discussion

“T-cadherin binds clinically important HMW multimer adiponectin with high affinity (Fukuda et al., 2017) and mediates adiponectin-induced exosome biogenesis and ceramide efflux to exosomes (Obata et al., 2018). […] These findings support Tcadherin mediating adiponectin functions as a receptor for native HMW multimer adiponectin (Kita et al., 2019).”

2) Along the same line, it is unclear whether T-cadherin might behave as a typical receptor protein for adiponectin. For example, typical receptors are saturated by ligands. However, provided data (Figures 1, 2 and 3) did not show any saturation pattern nor dose response. This issue needs to be properly addressed by experimental data.

We appreciate the valuable comment. We demonstrated the saturable binding of native adiponectin in serum. The binding isotherm obtained is consistent with the high-affinity interaction we reported previously using purified HMW adiponectin with purified T-cadherin (Fukuda et al., 2017). The results obtained were shown in Figure 1D. The following sentences were added.

Results and Discussion

“The dose-response study revealed the specific and saturable binding of native adiponectin in serum to cells expressing T-cadherin (Figure 1E).”

3) The authors demonstrated that native adiponectin bound to T-cadherin. Have you compared the binding affinity with native and recombinant adiponectin proteins to three potential receptors? If there is any difference, it needs to be described and discussed.

We appreciate the valuable comment. In the revised manuscript, we compared the binding of native adiponectin in serum, purified native adiponectin, and recombinant full-length adiponectin produced in HEK293 cells. The results obtained indicated that only cells expressing T-cadherin and not the other receptors bound these preparations of adiponectin. However, recombinant adiponectin with a lower amount of HMW multimer adiponectin showed weak binding at a physiological concentration (20 μg/mL) of adiponectin.

The results obtained were shown in Figure 1B and 1D. The following sentences were added.

Results and Discussion

“We directly examined mouse serum as the ligand solution, including the most native adiponectin, purified adiponectin from mouse serum (Fukuda et al., 2017), and full-length recombinant adiponectin produced in HEK293 cells. […] Recombinant adiponectin contained a lower amount of HMW multimer adiponectin than mouse serum and purified adiponectin from serum (Figure 1B).”

Results and Discussion

“The treatment of cells with different preparations of adiponectin at 4°C for 1 hr resulted in the binding of prepared adiponectin only to cells expressing mouse T-cadherin (Figure 1D). Mouse serum and purified adiponectin showed similar binding, whereas recombinant adiponectin containing a lower amount of 6-mer and the HMW multimer exhibited markedly weaker binding (Figure 1D).”

Once again, we are sincerely grateful to reviewer #2 for the constructive suggestions, which have improved the quality of the revised manuscript.

Reviewer #3:

In spite of the identification of adiponectin quite a number of years ago, the nature of its receptors remains controversial. The rationale for the current study is that the major criterion for evaluating a potential receptor should be that it binds native adiponectin and not just recombinant product. This seems quite reasonable and therefore the strength of the paper is that it seeks to accomplish that. The weakness is that the nature of the protocol involves performing the binding study on a very complex, on might say "dirty" system, in which it is challenging to infer the actual biochemical even being measured.

We appreciate the valuable comment. We compared the binding of native adiponectin in serum, purified native adiponectin, and recombinant full-length adiponectin produced in HEK293 cells. The results obtained indicated that only cells expressing T-cadherin and not the other receptors bound these preparations of adiponectin. However, recombinant adiponectin with lower amounts of HMW multimer adiponectin showed weak binding at a physiological concentration (20 μg/mL) of adiponectin. These results were shown in Figure 1B and 1D. We added the following sentences.

Results and Discussion

“We directly examined mouse serum as the ligand solution, including the most native adiponectin, purified adiponectin from mouse serum (Fukuda et al., 2017), and full-length recombinant adiponectin produced in HEK293 cells. […] Recombinant adiponectin contained a lower amount of HMW multimer adiponectin than mouse serum and purified adiponectin from serum (Figure 1B).”

Results and Discussion

“The treatment of cells with different preparations of adiponectin at 4°C for 1 hr resulted in the binding of prepared adiponectin only to cells expressing mouse T-cadherin (Figure 1D). Mouse serum and purified adiponectin showed similar binding, whereas recombinant adiponectin containing a lower amount of 6-mer and the HMW multimer exhibited markedly weaker binding (Figure 1D).”

Furthermore, we would like to emphasize that the ligand in serum is the most natural ligand that maintains its ability to interact with the physiological binding partner. Numerous constituents, including a large number of proteins, exert adequate blocking effects to investigate whether the interaction between the physiological ligand and binding protein is specific. Moreover, the use of a recombinant ligand may make the experiment unreproducible because the same recombinant ligand cannot be made at another site. The ligand-receptor interaction in a more purified system can have mean if the ligand can bind with the receptor in a more physiological situation like in serum as our experiment The results of our new experiment indicate that recombinant adiponectin has limited affinity to T-cadherin due to the presence of less of the HMW form.

At a minimum, the authors' need to be much more precise in their language and judicious in what they claim the data show. For example, the authors state "These results clearly indicate that T-cadherin can bind native adiponectin." This is a gross overinterpretation of this experiment. What the authors show is that overexpression of T-cadherin in cells allows them to bind increased amounts of adiponectin. There is no information to indicated that the adiponectin is binding directly to T-cadherin or, for that matter, that T-cadherin is involved in the binding of hormone. This interpretation might be plausible, but it is far from proven.

We appreciate the valuable comment. The revised manuscript was proofread by a native English speaker.

Since we previously reported a serum-derived purified APN interaction with purified Fc-fusion Tcadherin (JBC, 2017), the statement “These results clearly indicate that T-cadherin can bind native adiponectin”was replaced as follows.

Results and Discussion

“We previously reported that purified recombinant T-cadherin bound purified adiponectin with an affinity of KD=1.0 nM (Fukuda et al., 2017). The present results showed the saturable binding of native adiponectin in serum to cells expressing T-cadherin, which is consistent with previous findings (Fukuda et al., 2017).”

The authors do demonstrate that the binding is concentration dependent, which is important for true receptor binding, but they do not establish saturability, which is essential to arguing the physiological relevance of the event being measured. Thus, it would add to the weight of the argument of they could show a full binding curve up to the point of saturability. It is relatively straightforward to vary expression to make sure the receptor level is low enough that it can be saturated with the adiponectin levels present in serum.

We appreciate the valuable comment. We demonstrated the saturable binding of native adiponectin in serum. The binding isotherm obtained is consistent with the high-affinity interaction we reported previously using purified HMW adiponectin with purified T-cadherin (Fukuda et al., 2017). The results obtained were shown in Figure 1E. The following sentences were added.

Results and Discussion

“The dose-response study revealed the specific and saturable binding of native adiponectin in serum to cells expressing T-cadherin (Figure 1E).”

[Editors’ note: the author responses to the re-review follow.]

Reviewer #1:

This paper provides data supporting the view that binding of adiponectin to cells is through T-cadherin rather than other purported receptor types. There are three issues that diminish the impact of the paper:

1) The first paragraph is an overly long "stream of consciousness" type of Introduction that should be heavily edited and organized into multiple paragraphs around the several discrete ideas.

We reorganized Introduction section according to your kind advice as follows.

“Adiponectin is a circulating factor that is secreted from adipocytes (Hu et al., 1996; Maeda et al., 1996; Nakano et al., 1996; Scherer et al., 1995). […] We herein demonstrated that native adiponectin in serum bound to cells expressing T-cadherin, but not to those expressing AdipoRs or calreticulin.”

2) The major impact of this study would be that bio-effects of adiponectin are mediated through T-cadherin, not the other putative receptors, but unfortunately no bio-effects are studied in these experiments. While the results are very interesting and certainly provocative, a definitive experiment on this key point showing that T-cadherin but not the other receptors mediates important bio-effects is missing.

Thank you for pointing the most important point to be addressed. It is well known that adiponectin stimulates intracellular signaling cascades such as phospho-AMPK and phospho-p38. And the loss of AdipoRs was shown to decrease these signaling. One explanation will be the generation of low molecular weight form of adiponectin such as globular form after binding to T-cadherin. However, it was recently reported that the primary cellular function of AdipoR1 and AdipoR2 is to maintain membrane fluidity (Ruiz M et al., J Lipid Res. 2019 May;60(5):995-1004). Loss of them might disturb membrane fluidity and thereby decrease signaling. Another explanation may be intracellular signaling by HMW-adiponectin internalization through binding to T-cadherin without interaction with AdipoRs. In future studies, we will carefully examine whether HMW-adiponectin stimulates such signaling or not and whether endocytosis of adiponectin through T-cadherin is required or not.

3) The cell biology/microscopy data are too limited. Multiple fields should be shown and appropriate controls with null signal. The data should be quantified from many fields as well.

We appreciate the valuable comment. The fluorescent microscopy data (Figure 2H) only indicate that the tagged receptors are correctly expressed, together with Western blotting data (Figure 2E). At this moment, it seems kind of reasonable to us that quantification by microscopy data may not be required because we have more accurate quantified data by Western blots.

Reviewer #2:

In this work, Kita et al. have demonstrated that native adiponectin would exclusively bind to T-cadherin. With cell culture model, they showed that serum or purified adiponectin preferentially bound to T-cadherin but not to AdipoR nor Calreticulin. Although they provided more data and tried to strengthen Discussion part in the resubmitted manuscript, it still has technical limitations to reach the firm conclusion.

1) Although it has been reported that adiponectin binds to AdipoRs, calreticulin, and T-cadherin, they argued that the major binding protein for native adiponectin is T-cadherin. If the binding affinity of native adiponectin to adipoRs or calreticulin would be much weaker than T-cadherin, it seems that the experimental condition in this study might be too harsh to detect weak interaction with others. They have to adopt alternative methods to affirm the extent of protein interactions. Along the same line, they need to measure/compare binding affinity of recombinant adiponectin with other proteins including AdipoR and Calreticulin.

Thank you for pointing an important discussion point in this study. As discussed in the Results and Discussion section (ninth paragraph), in the case of calreticulin, the interaction with adiponectin was evidenced by a neutralizing antibody treatment inhibiting the adiponectin interaction with cells expressing calreticulin (Takemura et al., 2007). So, possibilities of the requirement of calreticulin for adiponectin binding such as the requirement of some accessory protein may exist. In the case of AdipoRs, AdipoR was discovered by expression cloning, as discussed in the Results and Discussion section (eighth paragraph). Expression cloning requires repeated washing steps before separation by FACS. Therefore, the overexpression of AdipoR is expected to promote firm ligand binding. The initial discovery of AdipoRs also indicated that HEK293 cells overexpressing AdipoRs bound E. coli recombinant globular adiponectin (Yamauchi et al., 2003). Therefore, difficulties are associated with speculating about the much weaker binding affinity or requirement for some “accessory” proteins to confer adiponectin binding activity to AdipoRs. Rather, HMW-adiponectin may not bind to AdipoRs, but the globular form may do.

2) To convince that native adiponectin exclusively binds to T-cadherin, it is crucial to provide positive control data.

We appreciate the valuable comment. We previously reported and established HMW-adiponectin binding to T-cadherin in several ways including Biacore analysis and loss of tissue accumulation of adiponectin in T-cadherin KO mice (Fukuda et al., 2017, Matsuda et al., 2015). Therefore in this sense, T-cadherin can be thought of as positive control. Based on nearly the same amounts of receptors expressed on the cell surface as judged by PA-tag expression outside of the cells, native adiponectin bound to the cells expressing T-cadherin but not to the cells expressing AdipoR1.

3) It has been suggested to examine intracellular signaling cascades such as phospho-AMPK and phospho-p38 upon the interaction between native adiponectin and potential receptors.

Thank you for pointing the most important point to be addressed. It has been shown that adiponectin stimulates intracellular signaling cascades such as phospho-AMPK and phospho-p38. And the loss of AdipoRs decreases these signaling. One explanation will be the generation of low molecular weight form of adiponectin such as globular form after binding to T-cadherin. However, it was recently reported that the primary cellular function of AdipoR1 and AdipoR2 is to maintain membrane fluidity (Ruiz M et al., J Lipid Res. 2019 May;60(5):995-1004). Loss of them might disturb membrane fluidity and thereby decrease signaling. Another explanation may be intracellular signaling by HMW-adiponectin internalization through binding to T-cadherin without interaction with AdipoRs. In our future studies, we will carefully examine whether HMW-adiponectin stimulates such signaling or not and whether endocytosis of adiponectin through T-cadherin is required or not.

Reviewer #3:

I was ambivalent about this manuscript the first time around and the authors have only improved it with the additional experiments. In all honesty, this will never be the cleanest study and will not have the highest level of impact due to lack of mechanism, whatever the authors do to address the issues. However, I continue to believe that it brings up a valid point about the ambiguity of the true adiponectin receptor and the data are strong enough to provide a valid challenge to the dogma. On balance, I recommend acceptance.

Thank you very much for evaluating the significance of our study. Regarding the mechanism, how adiponectin input intracellular signaling, it must be the next important study. It is well known that adiponectin stimulates intracellular signaling cascades such as phospho-AMPK and phospho-p38. And the loss of AdipoRs decreases these signaling. One explanation will be the generation of low molecular weight form of adiponectin such as globular form after binding to T-cadherin. However, it was recently reported that the primary cellular function of AdipoR1 and AdipoR2 is to maintain membrane fluidity (Ruiz M et al., J Lipid Res. 2019 May;60(5):995-1004). Loss of them might disturb membrane fluidity and thereby decrease signaling. Another explanation may be intracellular signaling by HMW-adiponectin internalization through binding to T-cadherin without interaction with AdipoRs. In our future studies, we will carefully examine whether HMW-adiponectin stimulates such signaling or not and whether endocytosis of adiponectin through T-cadherin is required or not.

Author details

1. Shunbun Kita

   1. Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan
   2. Department of Adipose Management, Graduate School of Medicine, Osaka University, Osaka, Japan

   Contribution

   Conceptualization, Resources, Software, Formal analysis, Funding acquisition, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing, Performed biochemical and cellular experiments

   For correspondence

   shunkita@endmet.med.osaka-u.ac.jp

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8937-0053

2. Shiro Fukuda

   Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan

   Contribution

   Resources, Data curation, Software, Validation, Visualization, Methodology, Writing—review and editing, Cloned the cDNAs of adiponectin receptors

   Competing interests

   No competing interests declared

3. Norikazu Maeda

   1. Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan
   2. Department of Metabolism and Atherosclerosis, Graduate School of Medicine, Osaka University, Osaka, Japan

   Contribution

   Data curation, Supervision, Funding acquisition, Writing—review and editing

   Competing interests

   No competing interests declared

4. Iichiro Shimomura

   Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan

   Contribution

   Data curation, Supervision, Funding acquisition, Project administration, Writing—review and editing

   Competing interests

   No competing interests declared

• 1,451

  Page views

• 256

  Downloads

• 21

  Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.