Spenito-dependent metabolic sexual dimorphism intrinsic to fat storage cells

Metabolism in males and females is distinct. Differences are usually linked to sexual reproduction, with circulating signals (e.g. hormones) playing major roles. By contrast, sex differences prior to sexual maturity and intrinsic to individual metabolic tissues are less understood. We analyzed Drosophila melanogaster larvae and find that males store more fat than females, the opposite of the sexual dimorphism in adults. We show that metabolic differences are intrinsic to the major fat storage tissue, including many differences in the expression of metabolic genes. Our previous work identified fat storage roles for Spenito (Nito), a conserved RNA-binding protein and regulator of sex determination. Nito knockdown specifically in the fat storage tissue abolished fat differences between males and females. We further show that Nito is required for sex-specific expression of the master regulator of sex determination, Sex-lethal (Sxl). “Feminization” of fat storage cells via tissue-specific overexpression of a Sxl target gene made larvae lean, reduced the fat differences between males and females, and induced female-like metabolic gene expression. Altogether, this study supports a model in which Nito autonomously controls sexual dimorphisms and differential expression of metabolic genes in fat cells in part through its regulation of the sex determination pathway. Total RNA from 50 dissected sexed, third-instar-larval fat bodies was extracted from 50 third-instar FBs dissected from sexed larvae using 500uL of TRIzol (ambion Cat # 15596018cat number), volumes and purified using the Direct-zol Miniprep Plus kit digested with DNase I (Zymo Cat # R2072cat number). cDNA was generated with 500ng of RNA sequencing and library prep was performed at the University of Colorado Anschutz medical campus Genomics Core. Transcriptome analysis was performed using pseudo-alignment with salmon [PMID 28263959] using the D. melanogaster transcriptome (version dmel_r6.48_FB2022_05). DESeq2 [59] (version 1.40.1) was used for differential expression analysis. Pathway analysis was performed using gProfiler [44] (version 0.2.1).

两性的新陈代谢存在显著差异。这类差异通常与有性生殖相关，循环信号（如激素）发挥着核心调控作用。与之相对，性成熟前的性别差异以及个体代谢组织固有的性别差异，目前尚缺乏充分研究。我们以黑腹果蝇（Drosophila melanogaster）幼虫为研究对象，发现雄性幼虫的脂肪储存量高于雌性，这与成虫的性别二态性特征恰好相反。研究表明，代谢差异源自主要脂肪储存组织的固有特性，其中包括大量代谢基因的表达差异。

我们此前的研究已证实，保守型RNA结合蛋白兼性别决定调控因子Spenito（简称Nito）具有脂肪储存调控功能。在脂肪储存组织中特异性敲低Nito，可消除雌雄个体间的脂肪储存差异。我们进一步证实，Nito是性别决定主调控因子Sex-lethal（Sxl）实现性别特异性表达的必需因子。通过在脂肪储存组织中特异性过表达Sxl的靶基因，实现脂肪细胞的“女性化”，可使幼虫脂肪含量降低，缩小雌雄个体间的脂肪储存差异，并诱导出现雌性样的代谢基因表达谱。

综上，本研究支持如下模型：Nito通过调控性别决定通路，自主控制脂肪细胞中的性别二态性以及代谢基因的差异表达。本研究从经性别鉴定的三龄幼虫中分离得到50个脂肪体（FB），使用500 μL TRIzol（Ambion Cat # 15596018）提取总RNA，随后采用Direct-zol Miniprep Plus试剂盒，并经DNase I（Zymo Cat # R2072）消化处理完成RNA纯化。以500 ng总RNA反转录合成cDNA，文库制备工作由科罗拉多大学安舒茨医学校区基因组学核心实验室完成。转录组分析采用salmon [PMID 28263959] 进行伪比对分析，参考转录组为黑腹果蝇转录组（版本dmel_r6.48_FB2022_05）。差异表达分析采用DESeq2 [59]（版本1.40.1），通路富集分析则使用gProfiler [44]（版本0.2.1）。