Semaphorin 4B is an ADAM17-cleaved adipokine that inhibits adipocyte differentiation and thermogenesis

**Background:** ADAM17 (TACE) is a metalloprotease that sheds cell surface signaling molecules, playing key roles in inflammation and growth. Its role in metabolic homeostasis, particularly in adipose tissue, is poorly understood. Previous studies showed that whole-body ADAM17 knockout mice exhibit hypermetabolism and are protected from obesity, but the underlying mechanisms and specific substrates remained unknown. This study aimed to determine the impact of adipocyte-specific ADAM17 deletion on metabolic homeostasis and to identify the relevant substrate(s).

**Methods:** The authors generated adipocyte-specific ADAM17 knockout mice (Adam17^adipoqΔ/Δ^) by crossing Adam17^fl/fl^ mice with Adipoq-Cre mice. Mice were fed a standard chow or high-fat diet (HFD, 60% energy from fat) for 26 weeks. Metabolic phenotyping included calorimetry (Promethion system), glucose and insulin tolerance tests, serum lipid and insulin measurements, liver triglyceride quantification, and histopathology of adipose tissues and liver. Primary adipocyte cultures from inguinal and brown adipose tissue were used for ex vivo experiments. Proteomic analysis of the secretome was performed using hiSPECS (high-performance secretome protein enrichment with click sugars) to identify ADAM17 substrates. Sema4B shedding was confirmed by ELISA and western blot. Lentiviral overexpression of a truncated, soluble form of Sema4B (tSema4B) in immortalized brown adipocytes was used to study its functional effects. RNA sequencing was performed on these cells under basal and NE-stimulated conditions. Human adipose tissue samples from obese and non-obese subjects, including paired samples before and after bariatric surgery, were analyzed for gene expression.

**Key Results:** Adipocyte-specific ADAM17 knockout mice (KO) exhibited a hypermetabolic phenotype. On HFD, KO mice gained less weight (body mass significantly lower, p<0.0001) and had reduced fat mass (pooled fat mass significantly lower, p<0.0001) despite consuming more calories (mean daily food intake significantly higher, p<0.01). Energy expenditure, oxygen consumption, and CO2 production were significantly elevated in KO mice (ANCOVA with body weight as covariate, p<0.05). KO mice showed increased BAT thermogenesis (interscapular temperature significantly higher, p<0.01) and elevated expression of thermogenic genes (Ucp1, Pgc1α, Prmd16, Dio2) in BAT (p<0.05). KO mice were protected from HFD-induced hepatosteatosis (liver triglyceride content significantly lower, p<0.01), dyslipidemia (total cholesterol and LDL-cholesterol significantly lower, p<0.05), and insulin resistance (fasting glucose and insulin significantly lower, HOMA-IR significantly lower, p<0.01; glucose tolerance and insulin tolerance significantly improved, p<0.05). Proteomic analysis identified Semaphorin 4B (Sema4B) as a novel ADAM17 substrate whose shedding was significantly reduced in KO adipocyte secretomes (p<0.05). NE stimulation increased Sema4B shedding in WT but not KO adipocytes, and this was blocked by the metalloprotease inhibitor BB94. Overexpression of tSema4B in immortalized brown adipocytes significantly inhibited Ucp1 expression (p<0.001), lipolytic genes (Atgl, Hsl; p<0.01), and adipogenic genes (Pparγ, C/ebpα; p<0.05). RNA sequencing revealed that tSema4B represses genes involved in brown adipocyte differentiation, fatty acid import, storage, and beta-oxidation, while upregulating extracellular matrix genes. In human adipose tissue, PLXNB2, NRP2, ADAM17, and iRHOM2 mRNA levels were elevated in obesity and decreased after bariatric surgery-induced weight loss.

**Clinical Implications:** This study identifies a novel ADAM17-Sema4B axis that negatively regulates adipocyte differentiation, thermogenesis, and lipid catabolism. Targeting this pathway, for example by inhibiting Sema4B signaling or enhancing its clearance, could represent a new therapeutic strategy for obesity and its metabolic complications. The findings also highlight the importance of ADAM17 in adipose tissue beyond its well-known roles in inflammation, and suggest that specific branches of ADAM17 biology (e.g., Sema4B shedding) may be targeted to avoid the toxicity associated with broad ADAM17 inhibition.