Microenvironments support the maintenance of stem cells and the growth of tumors through largely unknown mechanisms. While cell-autonomous chromatin modifications have emerged as important determinants for self-renewal and differentiation of stem cells, a role for non-cell autonomous epigenetic contributions is not well established. Here, we genetically ablated the chromatin modifier Sin3a in fetal Sertoli cells, which partly comprise the niche for male germline stem cells, and investigated its impact on spermatogenic cell fate and teratoma formation in vivo. Sertoli cell-specific Sin3a deletion resulted in the formation of few undifferentiated spermatogonia after birth while initially maintaining spermatogenic differentiation. Stem cell-associated markers Plzf, Gfra1, and Oct4 were downregulated in the mutant fetal gonad, while Sertoli cell markers Steel and Gdnf, which support germ cells, were not diminished. Following birth, markers of differentiating spermatogonia, Kit and Sohlh2, exhibited normal levels, but chemokine signaling molecules CXCL12/SDF1 and CXCR4, expressed in Sertoli cells and germ cells, respectively, were not detected. In the juvenile, mutant testes exhibited a progressive loss of differentiating spermatogonia and a block in spermatid elongation, followed by extensive germ cell degeneration. Sertoli cell-specific Sin3a deletion also suppressed teratoma formation by fetal germ cells in an in vivo transplantation assay. We conclude that the epigenome of Sertoli cells influences the establishment of a niche for germline stem cells, as well as for tumor initiating cells.

The inhibitory G protein alpha subunit, Gαz, is an important modulator of beta-cell function. Full-body Gαz-null mice are protected from hyperglycemia and glucose intolerance after long-term high-fat diet (HFD) feeding. In this study, at a time point in the feeding regimen where wild-type mice are only mildly glucose intolerant, transcriptomics analyses reveal islets from HFD-fed Gαz KO mice have a dramatically altered gene expression pattern as compared to WT HFD-fed mice, with entire gene pathways not only being more strongly up- or down-regulated vs. control-diet fed groups, but actually reversed in direction. Genes involved in the “Pancreatic Secretion” pathway are the most strongly differentially regulated: a finding that correlates with enhanced islet insulin secretion and decreased glucagon secretion at study end. The protection of Gαz-null mice from HFD-induced diabetes is β-cell autonomous, as β-cell-specific Gαz-null (βKO) mice phenocopy the full-body knockouts. The glucose-stimulated and incretin-potentiated insulin secretion response of islets from HFD-fed βKO mice is significantly improved as compared to islets from HFD-fed wild-type controls, which, along with no impact of Gαz loss or HFD feeding on beta-cell proliferation or surrogates of beta-cell mass supports a secretion-specific mechanism. Gαz is coupled to the Prostaglandin EP3 receptor in pancreatic beta-cells. We confirm the EP3γ splice variant has both constitutive and agonist-sensitive activity to inhibit cyclic AMP production and downstream β-cell function, with both activities being dependent on the presence of beta-cell Gαz.

One complication to comparing β-cell function among islet preparations, whether from genetically identical or diverse animals or human organ donors, is the number of islets required per assay. Islet numbers can be limiting, meaning that fewer conditions can be tested; other islet measurements must be excluded; or islets must be pooled from multiple animals/donors for each experiment. Furthermore, pooling islets negates the possibility of performing single-islet comparisons. Our aim was to validate a 96-well plate-based single islet insulin secretion assay that would be as robust as previously published methods to quantify glucose-stimulated insulin secretion from mouse and human islets. First, we tested our new assay using mouse islets, showing robust stimulation of insulin secretion 24 or 48 h after islet isolation. Next, we utilized the assay to quantify mouse islet function on an individual islet basis, measurements that would not be possible with the standard pooled islet assay methods. Next, we validated our new assay using human islets obtained from the Integrated Islet Distribution Program (IIDP). Human islets are known to have widely varying insulin secretion capacity, and using our new assay we reveal biologically relevant factors that are significantly correlated with human islet function, whether displayed as maximal insulin secretion response or fold-stimulation of insulin secretion. Overall, our results suggest this new microplate assay will be a useful tool for many laboratories, expert or not in islet techniques, to be able to precisely quantify islet insulin secretion from their models of interest.