The progression of metabolic diseases such as obesity and type 2 diabetes are driven by secreted proteins and peptides, acting in autocrine, paracrine, and/or endocrine pathways. Within the mediobasal hypothalamus (MBH), a crucial regulator of metabolism, peptide and protein secretion is orchestrated in response to nutritional status and obesity. While research has primarily focused on MBH-secreted neuropeptides derived from pro-opiomelanocortin (POMC), the broader landscape of MBH secretory molecules remains largely uncharacterised.
To address this knowledge gap, we have developed a pioneering methodology for identifying the secretory peptidome and proteome of the MBH in conscious, freely-behaving mice. This involves the precise placement of custom-built ultrabroad-capture microdialysis probes (molecular weight cut-off <1,000,000Da) and real-time, µL-resolution sampling for unbiased peptide and protein identification via LC-MS/MS.
We surgically implanted guide cannulas above the MBH in male C57Bl/6 mice fed either a high-fat diet or standard chow diet for up to 12 weeks. Mice were fasted overnight, and microdialysis probes were inserted into the MBH to collect dialysate for 2 hours in the fasted state. Subsequently, mice were allowed ad libitum access to food for 2 hours, during which fed dialysate was collected. Tandem mass tag labelling coupled with mass spectrometry-based proteomics/peptidomics enabled us to comprehensively uncover the MBH secretome.
Analysis using existing proteome databases identified thousands of proteins and peptides within the MBH secretome. Notably, our peptidomic analysis revealed that fasting suppressed POMC secretion in chow mice, which was dysregulated in obesity. This corroborates previous findings, reaffirming the sensitivity and relevance of our method. To compliment this analysis, we used our newly-developed long-read RNAseq-based proteogenomics workflow to identify unique, unannotated proteins/peptides regulated by fasting and/or obesity. Collectively, our innovative discovery approach has shed light on the intricate secretory profile of the MBH, and holds promise for identifying novel therapeutic targets for metabolic diseases.