Fig 2: Semaphorin 4B negatively regulates adipogenesis and thermogenesis. (A–C), mRNA expression of the thermogenic (A), lipolytic (B), and adipogenic (C) genes in immortalized primary adipocytes transduced with empty vector or truncated Sema4B (tSema4B) and treated with or without NE. (n = 6). (D–F), mRNA expression of the thermogenic (D), lipolytic (E), and adipogenic (F) genes in primary adipocytes treated with or without recombinant tSema4B and subsequently treated with or without NE (n = 5). (G), Volcano plots showing differentially expressed genes in immortalized primary brown adipocytes transduced with tSema4B or empty vector (Plex) and a list of some upregulated and downregulated genes by tSema4B (n = 3). (H), Volcano plots showing differentially expressed genes in immortalized primary brown adipocytes transduced with tSema4B or empty vector (Plex) in response to NE and a list of some upregulated and downregulated genes by tSema4B in response to NE (n = 3). (I), Heat map of upregulated and downregulated genes by tSema4B in naïve and NE-stimulated cells according to the GO:term biological process. (J), Oil red staining and quantification of oil red absorbance in differentiated immortalized primary brown adipocytes transduced with empty vector or truncated Sema4B (tSema4B). (K), Glycerol levels (pg/mL) in the medium of the cells indicated above under NE exposure or unstimulated conditions (L), Immunoblots of the phosphorylated (active) form of the lipolytic enzyme, hormone sensitive lipase (HSL) and the central lipolytic enzyme adipose triglyceride lipase (ATGL) in immortalized primary adipocytes transduced with empty vector or truncated Sema4B (tSema4B) with or without stimulation with NE. A p97 immunoblot is included as loading control. Results presented as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 3: Semaphorin 4B is a novel adipokine and an ADAM17 substrate. (A), Schematic showing how the potential ADAM17 substrates were arrived at. Proteins denoted by grey circles detected by the mass spec at a P value of >0.05 were excluded, as were proteins whose levels were elevated in ADAM17 KO secretomes (denoted in blue circles). Proteins whose levels were downregulated in ADAM17 KO secretomes are denoted in red circles while the small subset of these that fulfill the characteristics of known ADAM17 substrates (TM1, TM2 or GPI-anchored [63]) are denoted by green circles. (B–C), Volcano plot depicting proteins detected in the secretome of ADAM17 KO vs WT inguinal (B) or brown (C) primary adipocytes. Proteins with log2 difference greater than or less than zero are detected at higher or lower levels, respectively in conditioned medium from ADAM17 KO adipocytes. Proteins above the horizontal cut-off (-Log pvalue = 1.3) are significantly detected at higher or lower levels in conditioned medium from ADAM17 KO adipocytes (n = 5). (D–F), Sema4B levels quantified by ELISA in conditioned media from WT brown primary adipocytes treated with or without 10 μM of the metalloprotease inhibitor, batimastat (BB-94) and 2 μM norepinephrine (NE) (D), WT and ADAM17 KO primary inguinal (E) and brown (F) adipocytes with or without stimulation with 2 μM of NE for 10 h (n = 3). (G), Immunoblot of Sema4B in conditioned media from WT and ADAM17 KO Hek293 cells transfected with full-length Sema4B plasmid. Cells were either untreated or treated with 1 μM phorbol myristate acetate (PMA) and deglycosylated using PNGAse. Immunoblots for tubulin serve as a loading control. (n = 4). (H–J), mRNA levels of Sema4b and its receptors; Plxnb1 & 2, Nrp1 & 2 in the interscapular BAT (H), inguinal WAT (I), and epididymal WAT (J) of lean and HFD-induced obese mice (n = 4). (K–M), mRNA levels of Sema4b and its receptors Plxnb1 & 2, Nrp1 & 2 in inguinal (K) and brown (L) primary adipocytes treated without or with 2 μM NE (n = 5), and in the interscapular BAT (M) of mice exposed to different ambient temperatures; thermoneutrality (30 °C) (10 days), room temperature (22 °C), acute cold exposure (4° S) (4 °C for 6 h), and chronic cold exposure (4° L) (4 °C for 10 days) (n = 6). Results presented as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)