Purified Somatotrope Populations Include Cells with Multihormonal Potential That Produce GH, TSH and Prolactin in Response to Leptin Signals
Authors: Syed Mohsin , Akhter Noor , Allensworth‐James Melody , Odle Angela , Haney Anessa , Beneš Helen , MacNicol Melanie , MacNicol Angus , Childs Gwen 
Published: November 2023

Leptin signaling to anterior pituitary somatotropes is required to maintain their function and to prevent growth hormone deficiency (GHD). In order to determine the mechanisms by which leptin signaling regulates somatotrope function, we added a floxed tdTomato‐eGFP Cre‐reporter transgene to lines bearing Cre‐recombinase driven by the rGH promoter and produced Leptin receptor‐wild type (Lepr‐wt) and Lepr‐null, green fluorescent (eGFP+) somatotropes. All non‐Cre‐bearing cells (non‐somatotropes) fluoresce red with tdTomato. Whereas 98% of eGFP+ cells contained GH in control (Lepr‐wt) mice, only 20% of eGFP+ somatotrope Lepr‐null cells stored GH. Thus, the introduction of 4 floxed alleles did not compromise the efficiency of Cre‐recombinase as the reduction in GH protein levels was similar to that seen in non‐fluorescent somatotrope Lepr‐null deletion mutants. Fluorescence activated cell sorting (FACS) purification successfully generated sufficient eGFP+ somatotropes from one pituitary (250,000–350,000 cells in females and 100,000–150,000 cells in males) for EIAs of all pituitary hormones by the Millipore Mouse kit (diluted 1:2.5‐fold) analyzed with the Luminex platform. Over 98% of eGFP+ Lepr‐wt somatotropes contained GH and the pure somatotrope fraction contained 80% of the total recovered GH and 400% more GH than the Lepr‐wt tdTomato fraction. Pure somatotropes from Lepr‐null mutants showed a significant, 50% reduction in GH in the eGFP fraction. As expected, the tdTomato (non‐somatotrope) fraction contained >90% of luteinizing hormone (LH), follicle stimulating hormone (FSH) and adrenocorticotropin (ACTH) levels; somatotrope Lepr‐null deletion mutants showed no reductions in these hormones in either eGFP or tdTomato fractions. Surprisingly, thyroid stimulating hormone (TSH) levels were high in pure eGFP+ somatotropes from Lepr‐wt control females (24% of the total TSH) and there was a significant, 60% reduction in TSH levels in pure eGFP+ Lepr‐null somatotropes from females. Furthermore, we recovered 93–97% of the total prolactin levels in pure eGFP+ wt somatotropes from females or males, while prolactin levels were reduced by 50–60% in pure Lepr‐null eGFP+ somatotropes from both sexes. Immunolabeling showed that 64% (females) or 50% (males) of pure wt somatotropes expressed prolactin compared with only 8–15% of the non‐somatotrope, tdTomato cells. Prolactin immunolabeling was significantly reduced to 22% of eGFP+ Lepr‐null mutant somatotropes only in females. Thus, these novel models allow the purification of somatotropes from a single mouse, providing sufficient protein and mRNA for complex studies of signaling to gene expression. In this initial study of these models, we show that pure somatotropes include an unexpectedly high proportion of multihormonal cells, suggesting that the somatotrope population may include progenitor cells of the Pit1/Pou1f1 lineage, an important transcription factor for GH, TSH and prolactin. Parallel qPCR assays show a significant decrease in Gh, Tsh, and Prl mRNA levels in pituitary mRNA extracts from somatotrope Lepr‐null female mice. These qPCR assays are being repeated with mRNA fractions from pure wt and mutant somatotrope fractions. Collectively, these discoveries about the decline in expression of thesel three pituitary hormones in pure Lepr‐null somatotropes suggest that leptin may play a broad role in the maintenance of cells that derive from the Pit1‐dependent lineage.Support or Funding InformationNIH R03 HD059066, core facilities of the Center for Translational Neuroscience funded by IDeA award P20 GM103425, and the Sturgis Foundation.