SUMMARY: Mitochondria provide a variety of metabolites, in addition to ATP, to meet cell-specific needs. One such metabolite is phosphoenolpyruvate (PEP), which contains a higher-energy phosphate bond than ATP and has diverse biological functions. However, how mitochondria-generated PEP is delivered to the cytosol and fulfills cell-specific requirements remains elusive. Here, we show that SLC25A35 regulates mitochondrial PEP efflux and glyceroneogenesis in lipogenic cells that utilize the pyruvate-to-PEP bypass. Reconstitution and structural studies demonstrated PEP transport by SLC25A35 in a pH gradient-dependent manner. Loss of SLC25A35 in adipocytes impaired the conversion of mitochondrial PEP into glycerol-3-phosphate, thereby reducing glycerolipid synthesis. Significantly, hepatic inhibition of SLC25A35 in obese mice alleviated steatosis and improved systemic glucose homeostasis. Together, these results suggest that mitochondria facilitate glycerolipid synthesis by providing PEP via SLC25A35, offering lipogenic mitochondria as a target to limit glycerolipid synthesis, a pivotal step in the pathogenesis of hepatic steatosis and Type 2 diabetes.

During sexual reproduction, the freshwater ciliate Tetrahymena thermophila sheds membrane-bound vesicles into the extracellular environment (cEMVs: ciliary extracellular micro-vesicles). We provide evidence that 100 nm vesicles shed from the cilia of starved cells promote mating between cells of complementary mating types. A proteomic analysis revealed that these EMVs are decorated with mating-type proteins expressed from the MAT locus, proteins that define a cell’s sex (one of seven). Once the mating junction is established between cells, smaller 60 nm vesicles (junction vesicles) appear within the extracellular gap that separates mating partners. Junction vesicles (jEMVs) may play a role in remodeling the mating junction through which gametic pronuclei are exchanged. Evidence is presented demonstrating that cells must be able to internalize extracellular signals via some form of endocytosis in order to trigger conjugation. Finally, an evolutionarily conserved fusogen (Hap2) implicated in pore formation also appears necessary for jEMV processing. This system offers an excellent opportunity for studies on ectosome shedding, intercellular signaling and shed vesicle uptake by macro-pinocytosis, as they relate to sexual reproduction in the ciliate Tetrahymena thermophila.

Micropeptides (microproteins) encoded by transcripts previously annotated as long noncoding RNA (IncRNAs) are emerging as important mediators of fundamental biological processes in health and disease. Here we applied two computational tools to identify putative micropeptides encoded by lncRNAs that are expressed in the human pancreas. We experimentally verified one such micropeptide encoded by a β-cell- and neural cell-enriched lncRNA TUNAR ( also known as TUNA, HI-LNC78 or LINC00617 ). We named this highly conserved 48-amino-acid micropeptide B eta cell- and N eural cell-regu lin (BNLN). BNLN contains a single-pass transmembrane domain and localized at the endoplasmic reticulum in pancreatic β-cells. Overexpression of BNLN lowered ER calcium levels, increased cytosolic calcium levels, and maintained ER homeostasis in response to high glucose challenge. To determine the physiological and pathological roles of BNLN, we assessed the BNLN expression in islets from mice fed with a high-fat diet and a regular diet, and found that BNLN is suppressed by diet-induced obesity (DIO). Conversely, overexpression of BNLN elevated glucose-stimulated insulin secretion in INS-1 cells. Lastly, BNLN overexpression enhanced insulin secretion in islets from lean and obese mice as well as from humans. Taken together, our study provides the first evidence that lncRNA-encoded micropeptides play a critical role in pancreatic β-cell function and provides a foundation for future comprehensive analyses of micropeptide function and pathophysiological impact on diabetes.