Calsyntenin-3b constrains lipid droplet expansion to promote a multilocular adipocyte phenotype and thermogenesis

Multilocular adipocytes are a hallmark of thermogenic adipose tissue, but the factors that enforce this cellular phenotype are unknown. Here, we show that an adipocyte-specific product of the Clstn3 locus (CLSTN3b) present only in placental mammals facilitates the rapid utilization of stored triglyceride by limiting lipid droplet (LD) size. CLSTN3b is an integral ER protein that localizes to ER-LD contact sites via a conserved hairpin domain. Mice lacking CLSTN3b have altered LD morphology and increased lipid accumulation in BAT, as well as heightened sensitivity to cold challenge, despite having no defect in adrenergic signaling. Conversely, forced expression of CLSTN3b promotes a multilocular LD phenotype in cultured cells and BAT and facilitates triglyceride utilization. Mechanistically, CLSTN3b associates with CIDE proteins and impairs their ability to transfer lipid between droplets, thereby limiting LD expansion. These findings define a molecular mechanism that maximizes LD surface area to facilitate lipid utilization in thermogenic adipocytes. RNA-seq was performed on brown adipose tissue (BAT) from 10 week old male WT and adipocyte-specific CLSTN3B knockout (AdC3KO) mice acclimated to thermoneutrality (30 C) for 2 weeks (n = 3, 3) and subsequently exposed to cold (4 C) for 6 hours (n = 4, 4). Total RNA was extracted using TRIzol and the RNeasy Mini Kit with on-column DNase I digestion (Qiagen). Library preparation and sequencing was done at GENEWIZ. Adapter and quality trimming of raw FASTQ files was performed using Trimmomatic. FastQC was used to analyze FASTQ files before and after trimming. Trimmed FASTQ files were aligned to GRCm38/mm10 using STAR. HTSeq-count was used to extract gene counts and differential gene expression analysis was performed using DESeq2.